Human serum albumin variants and uses thereof

ABSTRACT

A serum albumin variant, or functional fragment thereof, comprising one or more amino acid substitutions selected from (i) glycine, isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine, valine and leucine substituted for glutamine at position (522); (ii) valine substituted for alanine at position (552); and (iii) alanine, glutamic acid, histidine, serine, lysine and arginine substituted for glycine at position (572).

RELATED APPLICATION DATA

The present application claims priority from Australian PatentApplication No. 2017904211 entitled ‘Human serum albumin variant anduses thereof’ filed 18 Oct. 2017. The entire contents of which is herebyincorporated by reference.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicform. The entire contents of the Sequence Listing are herebyincorporated by reference.

FIELD

The present disclosure relates to human serum albumin variants and usesthereof.

BACKGROUND

Serum albumin is the most abundant, naturally occurring protein in humanplasma, with a major role in maintaining the osmotic pressure of blood,as well as in the transport of various substances in the blood stream.

Serum albumin is known to bind to a number of proteins in vivo,including the neonatal Fc receptor (FcRn) and this interaction is knownto be important for the plasma half-life of albumin FcRn is a membranebound protein, expressed in many cell and tissue types (e.g.,endothelial cells), and is constantly being internalised and recycled.Albumin does not bind to FcRn at neutral pH but once endocytosed albuminbinds to FcRn under the acidic conditions of the endosome. FcRn-boundalbumin is rescued from degradation and is recycled back to the cellsurface together with FcRn where, at physiological pH, it dissociatesfrom FcRn.

Albumin has a long plasma half-life of approximately 19 days. This longhalf-life has led to the use of albumin to extend the half-life ofpharmaceutical compounds. For example, albumin has been fused to humancoagulation Factor IX (FIX) resulting in extended half-life of FIX(IDELVION®). Albumin has also been conjugated to chemotherapeuticcompounds (such as paclitaxel) to increase drug half-life and drugaccumulation (e.g., Abraxane®).

Human serum albumin variants having one or more amino acid substitutionsthat result in improved binding to or affinity for FcRn compared tonatural serum albumin have been previously described (e.g.,WO2011051489).

However, it will be clear to the skilled person that there is anon-going need in the art for serum albumin variants with improvedpharmacokinetic properties, such as increased affinity for FcRn,increased plasma half-life and/or reduced clearance. There is also aneed in the art to develop methods for controlling the plasma half-lifeof drugs in the plasma to optimize drug dosing and accumulation.

SUMMARY

The present disclosure is based on the inventors' identification thatcertain amino acid substitutions in serum albumin improve or increaseits binding to the neonatal Fc receptor (FcRn). Serum albumin variantsthat have certain amino acid substitutions are capable of enhancedplasma half-life.

The inventors have determined that residues 522, 552 and 572 of SEQ IDNO: 1 are important for binding to the FcRn. Furthermore, the inventorshave identified that substitution at these residues with certain aminoacids increases or enhances binding to FcRn at acidic pH.

The findings by the inventors provide the basis for a serum albuminvariant comprising one or more amino acid substitutions at a residuecorresponding to amino acid 522, 552 or 572 of SEQ ID NO: 1. Thefindings by the inventors also provide the basis for methods fortreating a disorder, e.g., a bleeding disorder in a subject.

The present disclosure provides, a serum albumin variant, or functionalfragment thereof, comprising one or more amino acid substitutionsselected from the group consisting of:

-   -   (i) an amino acid selected from the group consisting of glycine,        isoleucine, lysine, methionine, phenylalanine, tryptophan,        tyrosine, valine and leucine substituted for glutamine at a        position corresponding to amino acid 522 of SEQ ID NO: 1;    -   (ii) valine substituted for alanine at a position corresponding        to amino acid 552 of SEQ ID NO: 1;    -   (iii) an amino acid selected from the group consisting of        alanine, glutamic acid, histidine, serine, lysine and arginine        substituted for glycine at a position corresponding to amino        acid 572 of SEQ ID NO: 1; and    -   (iv) combinations thereof.

In one example, the serum albumin variant, or functional fragmentthereof, comprises an amino acid selected from the group consisting ofglycine, isoleucine, lysine, methionine, phenylalanine, tryptophan,tyrosine, valine, and leucine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises glycine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises isoleucine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises lysine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises methionine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises phenylalanine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises tryptophan substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises tyrosine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises valine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises leucine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises alanine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises valine substituted for alanine at a positioncorresponding to amino acid 552 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises an amino acid selected from the group consisting ofalanine, glutamic acid, histidine, serine, lysine and argininesubstituted for glycine at a position corresponding to amino acid 572 ofSEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises alanine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises glutamic acid substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises histidine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises serine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises lysine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises arginine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises valine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, comprises an amino acid substitution as set out above at aposition corresponding to amino acid 522 of SEQ ID NO: 1, amino acid 552of SEQ ID NO: 1 and amino acid 572 of SEQ ID NO: 1.

In one example, the serum albumin variant, or functional fragmentthereof, optionally comprises one or more amino acid substitutions,deletions or insertions in addition to an amino acid substitution as setout above at a position corresponding to amino acid 522 of SEQ ID NO: 1,amino acid 552 of SEQ ID NO: 1 and amino acid 572 of SEQ ID NO: 1.Additional amino acid substitutions suitable for use in the presentdisclosure will be apparent to the skilled person and includenaturally-occurring substitutions and engineered substitutions such asthose described, for example, in WO2011051489.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure has improved pharmacokinetic propertiescompared to a serum albumin set forth in SEQ ID NO: 1. Pharmacokineticproperties of serum albumin will be apparent to the skilled person andinclude, for example, binding affinity to FcRn, plasma half-life and/orplasma clearance rate. Methods for determining pharmacokineticproperties of a serum albumin variant of the present disclosure will beapparent to the skilled person and/or described herein.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure binds with increased affinity to FcRn comparedto a serum albumin set forth in SEQ ID NO: 1. Methods for determiningthe affinity of the serum albumin variant, or functional fragmentthereof, to FcRn will be apparent to the skilled person and/or describedherein. In one example, the binding affinity of the serum albuminvariant, or functional fragment thereof, for FcRn is determined by flowcytometry. For example, CHO cells stably expressing the serum albuminvariant, or functional fragment thereof, are stained with alexa-488labeled FcRn/β2m (to detect target binding) and anti-myc-alexa 647 (todetect expression) at acidic (pH 5.5) and neutral (pH 7.4) pH andanalysed by flow cytometry. In one example, the affinity of the serumalbumin variant, or functional fragment thereof, to FcRn/β2m isdetermined by calculating mean fluorescence intensity relative to anunmodified serum albumin (e.g., as set forth in SEQ ID NO: 1). In oneexample, the affinity of the serum albumin variant, or functionalfragment thereof, is determined by biosensor analysis (e.g., using asurface plasmon resonance (SPR) assay). For example, the bindingaffinity (i.e., strength of interaction) of serum albumin variant, orfunctional fragment thereof, to immobilized FcRn is determined at pH 5.4and/or pH 7.4 and 37° C. In another example, the binding affinity (i.e.,strength of interaction) of immobilized serum albumin variant, orfunctional fragment thereof, to FcRn is determined at pH 5.4 and/or pH7.4 and 37° C. In one example, the affinity constant (K_(D)),dissociation constant (Kd) and association constant (Ka) are determined.For example, the affinity constant (K_(D)) is the ratio of thedissociation constant (Kd) and association constant (Ka) (i.e.,K_(D)=Kd/Ka).

In one example, the binding affinity is measured at acidic pH. Forexample, an acidic pH is a pH of less than about pH 6.0, such as aboutpH 5.9, or about pH 5.8, or about pH 5.7, or about pH 5.6, or about pH5.5, or about pH 5.4, or about pH 5.3, or about pH 5.2, or about pH 5.1,or about pH 5.0. In one example, the serum albumin variant, orfunctional fragment thereof, of the present disclosure binds withincreased affinity to FcRn at acidic pH compared to a serum albumin setforth in SEQ ID NO: 1. For example, the serum albumin variant, orfunctional fragment thereof, of the present disclosure binds withincreased affinity to FcRn at lower pH, e.g., about pH 6.0, tofacilitate binding in an endosome. In one example, the serum albuminvariant, or functional fragment thereof binds with increased affinity toFcRn at about pH 6.0 compared to its affinity at about pH 7.4, whichfacilitates the re-release of the serum albumin variant into bloodfollowing cellular recycling. The amino acid substitutions of thepresent disclosure are useful for extending the half-life of theprotein, by increasing FcRn-mediated recycling and thereby reducingclearance from the blood.

In one example, the level of binding of the serum albumin variant, orfunctional fragment thereof, of the present disclosure to FcRn isincreased at pH 5.4 as determined by cell-binding studies compared to aserum albumin set forth in SEQ ID NO: 1. In one example, the level ofbinding of the serum albumin variant, or functional fragment thereof, ofthe present disclosure to FcRn is increased at pH 5.4 as determined bycell-binding studies compared to its affinity at pH 7.4. In one example,the level of binding of the serum albumin variant, or functionalfragment thereof, of the present disclosure to FcRn is increased at pH5.4 as determined by SPR analysis compared to its affinity at pH 7.4. Inone example, the level of binding of the serum albumin variant, orfunctional fragment thereof, of the present disclosure to FcRn isincreased at pH 5.4 as determined by flow cytometry compared to itsaffinity at pH 7.4.

In one example, the level of binding of the serum albumin variant, orfunctional fragment thereof, of the present disclosure to FcRn isincreased by at least about 2 fold, or at least by about 4 fold, or atleast by about 5 fold, or at least by about 10 fold, or at least byabout 15 fold compared to a serum albumin set forth in SEQ ID NO: 1. Inone example, the level of binding of the serum albumin variant to theFcRn is increased by about 2 fold to about 5 fold compared to a serumalbumin set forth in SEQ ID NO: 1. For example, the level of binding ofthe serum albumin variant to the FcRn is increased by about 2 fold, orabout 2.25 fold, or about 2.5 fold, or about 2.75 fold, or about 3 fold,or about 3.25 fold, or about 3.5 fold, or about 3.75 fold, or about 4fold, or about 4.25 fold, or about 4.5 fold, or about 4.75 fold, orabout 5 fold compared to a serum albumin set forth in SEQ ID NO: 1. Inone example, the level of binding of the serum albumin variant to theFcRn is increased by about 2.4 fold, or about 2.8 fold, or about 3.1fold, or about 3.4 fold, or about 3.7 fold, or about 3.9 fold, or about4 fold, or about 4.8 fold compared to a serum albumin set forth in SEQID NO: 1.

In one example, the level of binding of the serum albumin variant to theFcRn is increased by about 5 fold to about 15 fold compared to a serumalbumin set forth in SEQ ID NO: 1. For example, the level of binding ofthe serum albumin variant to the FcRn is increased by about 5 fold, orabout 5.5 fold, or about 6 fold, or about 6.5 fold, or about 7 fold, orabout 7.5 fold, or about 8 fold, or about 8.5 fold, or about 9 fold, orabout 9.5 fold, or about 10 fold, or about 10.5 fold, or about 11 fold,or about 11.5 fold, or about 12 fold, or about 12.5 fold, or about 13fold, or about 13.5 fold, or about 14 fold, or about 14.5 fold, or about15 fold. In one example, the level of binding of the serum albuminvariant to the FcRn is increased by about 7.6 fold, or about 8.6 fold,or about 13.8 fold compared to a serum albumin set forth in SEQ ID NO:1.

In one example, the level of binding of the serum albumin variant to theFcRn is increased by about 15 fold to about 50 fold compared to a serumalbumin set forth in SEQ ID NO: 1. For example, the level of binding ofthe serum albumin variant to the FcRn is increased by about 15 fold, orabout 20 fold, or about 25 fold, or about 30 fold, or about 35 fold, orabout 40 fold, or about 45 fold, or about 50 fold compared to a serumalbumin set forth in SEQ ID NO: 1. In one example, the level of bindingof the serum albumin variant to the FcRn is increased by about 42 fold,or about 43 fold, or about 47 fold, or about 48 fold compared to a serumalbumin set forth in SEQ ID NO: 1.

In another example, the level of binding of the serum albumin variant tothe FcRn is increased by about 50 to about 100 fold compared to a serumalbumin set forth in SEQ ID NO: 1. For example, the level of binding ofthe serum albumin variant to the FcRn is increased by about 50 fold, orabout 55 fold, or about 60 fold, or about 65 fold, or about 70 fold, orabout 75 fold, or about 80 fold, or about 85 fold, or about 90 fold, orabout 95 fold, or about 100 fold compared to a serum albumin set forthin SEQ ID NO: 1. In one example, the level of binding of the serumalbumin variant to the FcRn is increased by about 57 fold, or about 58fold compared to a serum albumin set forth in SEQ ID NO: 1.

In a further example, the level of binding of the serum albumin variantto the FcRn is increased by about 100 fold to about 250 fold compared toa serum albumin set forth in SEQ ID NO: 1. For example, the level ofbinding of the serum albumin variant to the FcRn is increased by about100 fold, or about 110 fold, or about 120 fold, or about 130 fold, orabout 140 fold, or about 150 fold, or about 160 fold, or about 170 fold,or about 180 fold, or about 190 fold, or about 200 fold, or about 210fold, or about 220 fold, or about 230 fold, or about 240 fold, or about250 fold compared to a serum albumin set forth in SEQ ID NO: 1. In oneexample, the level of binding of the serum albumin variant to the FcRnis increased by about 180 fold, or about 240 fold compared to a serumalbumin set forth in SEQ ID NO: 1.

In one example, the level of binding of the serum albumin variant to theFcRn is increased by at least 250 fold compared to a serum albumin setforth in SEQ ID NO: 1. For example, the level of binding of the serumalbumin variant to the FcRn is increased by about 200 fold, or about 250fold, or about 300 fold, or about 350 fold, or about 400 fold, or about450 fold, or about 500 fold compared to a serum albumin set forth in SEQID NO: 1. In one example, the level of binding of the serum albuminvariant to the FcRn is increased by about 410 fold compared to a serumalbumin set forth in SEQ ID NO: 1.

In one example, serum half-life of the serum albumin variant isincreased compared to a serum albumin set forth in SEQ ID NO: 1. Forexample, the serum half-life of a serum albumin variant of the presentdisclosure is increased by at least about 1.5 fold compared to a serumalbumin set forth in SEQ ID NO: 1. In one example, the serum half-lifeof a serum albumin variant of the present disclosure is increased byabout 1.5 fold, or about 2 fold, or about 2.5 fold, or about 3 fold, orabout 3.5 fold, or about 4 fold, or about 4.5 fold, or about 5 fold, orabout 5.5 fold, or about 6 fold, or about 6.5 fold, or about 7 fold, orabout 7.5 fold, or about 8 fold, or about 8.5 fold, or about 9 fold, orabout 9.5 fold, or about 10 fold. In one example, the serum half-life ofa serum albumin variant of the present disclosure is increased by about5 days to about 10 days or more. For example, the serum half-life of aserum albumin variant of the present disclosure is increased by about 5days, or about 6 days, or about 7 days, or about 8 days, or about 9days, or about 10 days, or about 11 days, or about 12 days, or about 13days, or about 14 days, or about 15 days, or about 16 days, or about 17days, or about 18 days, or about 19 days, or about 20 days, or about 25days, or about 30 days, or about 35 days, or about 40 days, or about 45days, or about 50 days. Methods for determining the half-life of theserum albumin variant will be apparent to the skilled person and/ordescribed herein. In one example, the half-life of the serum albuminvariant is determined using an in vivo assay. In one example, the serumalbumin concentration is measured in an Enzyme-Linked ImmunosorbentAssay (ELISA) using human serum albumin specific antibodies. Forexample, the ELISA is performed using commercially available methods. Inanother example, serum albumin variant is injected intravenously intomice or cynomolgus monkeys and the plasma concentration is periodicallymeasured as a function of time. In one example, the plasma concentrationof serum albumin variant is measured at 3 minutes to 72 hours after theinjection. In one example, the plasma concentration of serum albuminvariant is measured up to 60 days after the injection. In one example,the serum albumin variant is radiolabelled. In one example, the in vivohalf-life of the serum albumin variant, or functional fragment thereof,is determined by calculating the clearance rate in beta-phase. In oneexample, the in vivo half-life of the serum albumin variant, orfunctional fragment thereof is compared to the in vivo half-life ofunmodified serum albumin (e.g., as set forth in SEQ ID NO: 1).

In one example, the rate of clearance (i.e., recycling and uptake) ofthe serum albumin variant is decreased compared to a serum albumin setforth in SEQ ID NO: 1. Methods for determining the rate of clearance(i.e., recycling and uptake) of the serum albumin variant will beapparent to the skilled person and/or described herein. In one example,confocal fluorescence microscopy is used to determine if the serumalbumin variant is recycled. For example, to determine if a serumalbumin variant is recycled, fluorescently labelled serum albuminvariant is incubated with cells expressing human FcRn receptor on thecell surface and visualised by confocal fluorescence microscopy.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises:

-   -   (i) an amino acid selected from the group consisting of glycine,        isoleucine, lysine, methionine, phenylalanine, tryptophan,        tyrosine, valine and leucine substituted for glutamine at a        position corresponding to amino acid 522 of SEQ ID NO: 1, and        valine substituted for alanine at position 552 of SEQ ID NO: 1;        or    -   (ii) an amino acid selected from the group consisting of        glycine, isoleucine, lysine, methionine, phenylalanine,        tryptophan, tyrosine, valine and leucine substituted for        glutamine at a position corresponding to amino acid 522 of SEQ        ID NO: 1, and an amino acid selected from the group consisting        of alanine, glutamic acid, histidine, serine, lysine and        arginine substituted for glycine at position 572 of SEQ ID NO:        1; or    -   (iii) valine substituted for alanine at a position corresponding        to amino acid 552 of SEQ ID NO: 1, and an amino acid selected        from the group consisting of alanine, glutamic acid, histidine,        serine, lysine and arginine substituted for glycine at position        572 of SEQ ID NO: 1; or    -   (iv) an amino acid selected from the group consisting of        glycine, isoleucine, lysine, methionine, phenylalanine,        tryptophan, tyrosine, valine and leucine substituted for        glutamine at a position corresponding to amino acid 522 of SEQ        ID NO: 1, valine substituted for alanine at position 552 of SEQ        ID NO: 1, and an amino acid selected from the group consisting        of alanine, glutamic acid, histidine, serine, lysine and        arginine substituted for glycine at position 572 of SEQ ID NO:        1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises an amino acid selected from thegroup consisting of glycine, isoleucine, lysine, methionine,phenylalanine, tryptophan, tyrosine, valine and leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,and valine substituted for alanine at position 552 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises an amino acid selected from thegroup consisting of glycine, isoleucine, lysine, methionine,phenylalanine, tryptophan, tyrosine, valine and leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,and an amino acid selected from the group consisting of alanine,glutamic acid, histidine, serine, lysine and arginine substituted forglycine at position 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises valine substituted for alanine at aposition corresponding to amino acid 552 of SEQ ID NO: 1, and an aminoacid selected from the group consisting of alanine, glutamic acid,histidine, serine, lysine and arginine substituted for glycine atposition 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises an amino acid selected from thegroup consisting of glycine, isoleucine, lysine, methionine,phenylalanine, tryptophan, tyrosine, valine and leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,valine substituted for alanine at position 552 of SEQ ID NO: 1, and anamino acid selected from the group consisting of alanine, glutamic acid,histidine, serine, lysine and arginine substituted for glycine atposition 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure additionally comprises tyrosine substitutedfor lysine at a position corresponding to amino acid 573 of SEQ ID NO:1.

In one example, a serum albumin variant, or functional fragment thereof,comprises an amino acid selected from the group consisting of glycine,isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine,valine and leucine substituted for glutamine at a position correspondingto amino acid 522 of SEQ ID NO: 1, and tyrosine substituted for lysineat a position corresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,comprises valine substituted for alanine at a position corresponding toamino acid 552 of SEQ ID NO: 1, and tyrosine substituted for lysine at aposition corresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,comprises an amino acid selected from the group consisting of alanine,glutamic acid, histidine, serine, lysine and arginine substituted forglycine at a position corresponding to amino acid 572 of SEQ ID NO: 1,and tyrosine substituted for lysine at a position corresponding to aminoacid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises an amino acid selected from thegroup consisting of glycine, isoleucine, lysine, methionine,phenylalanine, tryptophan, tyrosine, valine and leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,valine substituted for alanine at position 552 of SEQ ID NO: 1, andtyrosine substituted for lysine at a position corresponding to aminoacid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises an amino acid selected from thegroup consisting of glycine, isoleucine, lysine, methionine,phenylalanine, tryptophan, tyrosine, valine and leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,an amino acid selected from the group consisting of alanine, glutamicacid, histidine, serine, lysine and arginine substituted for glycine atposition 572 of SEQ ID NO: 1, and tyrosine substituted for lysine at aposition corresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises valine substituted for alanine at aposition corresponding to amino acid 552 of SEQ ID NO: 1, an amino acidselected from the group consisting of alanine, glutamic acid, histidine,serine, lysine and arginine substituted for glycine at position 572 ofSEQ ID NO: 1, and tyrosine substituted for lysine at a positioncorresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises an amino acid selected from thegroup consisting of glycine, isoleucine, lysine, methionine,phenylalanine, tryptophan, tyrosine, valine and leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,valine substituted for alanine at position 552 of SEQ ID NO: 1, an aminoacid selected from the group consisting of alanine, glutamic acid,histidine, serine, lysine and arginine substituted for glycine atposition 572 of SEQ ID NO: 1, and tyrosine substituted for lysine at aposition corresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises:

-   -   (i) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1; and/or    -   (ii) valine substituted for alanine at a position corresponding        to amino acid 552 of SEQ ID NO: 1; and/or    -   (iii) arginine substituted for glycine at a position        corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises valine substituted for alanine at aposition corresponding to amino acid 552 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises arginine substituted for glycine ata position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises:

-   -   (i) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1, and arginine        substituted for glycine at a position corresponding to amino        acid 572 of SEQ ID NO: 1; or    -   (ii) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1, and valine        substituted for alanine at a position corresponding to amino        acid 552 of SEQ ID NO: 1; or    -   (iii) valine substituted for alanine at a position corresponding        to amino acid 552 of SEQ ID NO: 1, and arginine substituted for        glycine at a position corresponding to amino acid 572 of SEQ ID        NO: 1; or    -   (iv) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1, and tyrosine        substituted for lysine at a position corresponding to amino acid        573 of SEQ ID NO: 1; or    -   (v) valine substituted for alanine at a position corresponding        to amino acid 552 of SEQ ID NO: 1, and tyrosine substituted for        lysine at a position corresponding to amino acid 573 of SEQ ID        NO: 1; or    -   (vi) arginine substituted for glycine at a position        corresponding to amino acid 572 of SEQ ID NO: 1, and tyrosine        substituted for lysine at a position corresponding to amino acid        573 of SEQ ID NO: 1; or    -   (vii) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1, valine        substituted for alanine at a position corresponding to amino        acid 552 of SEQ ID NO: 1, and arginine substituted for glycine        at a position corresponding to amino acid 572 of SEQ ID NO: 1;        or    -   (viii) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1, valine        substituted for alanine at a position corresponding to amino        acid 552 of SEQ ID NO: 1, and tyrosine substituted for lysine at        a position corresponding to amino acid 573 of SEQ ID NO: 1; or    -   (ix) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1, arginine        substituted for glycine at a position corresponding to amino        acid 572 of SEQ ID NO: 1, and tyrosine substituted for lysine at        a position corresponding to amino acid 573 of SEQ ID NO: 1; or    -   (x) leucine substituted for glutamine at a position        corresponding to amino acid 522 of SEQ ID NO: 1, valine        substituted for alanine at position 552 of SEQ ID NO: 1,        arginine substituted for glycine at a position corresponding to        amino acid 572 of SEQ ID NO: 1, and tyrosine substituted for        lysine at a position corresponding to amino acid 573 of SEQ ID        NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1, and argininesubstituted for glycine at a position corresponding to amino acid 572 ofSEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1, and valinesubstituted for alanine at a position corresponding to amino acid 552 ofSEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises valine substituted for alanine at aposition corresponding to amino acid 552 of SEQ ID NO: 1, and argininesubstituted for glycine at a position corresponding to amino acid 572 ofSEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1, and tyrosinesubstituted for lysine at a position corresponding to amino acid 573 ofSEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises valine substituted for alanine at aposition corresponding to amino acid 552 of SEQ ID NO: 1, and tyrosinesubstituted for lysine at a position corresponding to amino acid 573 ofSEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises arginine substituted for glycine ata position corresponding to amino acid 572 of SEQ ID NO: 1, and tyrosinesubstituted for lysine at a position corresponding to amino acid 573 ofSEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1, valinesubstituted for alanine at a position corresponding to amino acid 552 ofSEQ ID NO: 1, and arginine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1, valinesubstituted for alanine at a position corresponding to amino acid 552 ofSEQ ID NO: 1, and tyrosine substituted for lysine at a positioncorresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1, argininesubstituted for glycine at a position corresponding to amino acid 572 ofSEQ ID NO: 1, and tyrosine substituted for lysine at a positioncorresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises leucine substituted for glutamine ata position corresponding to amino acid 522 of SEQ ID NO: 1, valinesubstituted for alanine at position 552 of SEQ ID NO: 1, argininesubstituted for glycine at a position corresponding to amino acid 572 ofSEQ ID NO: 1, and tyrosine substituted for lysine at a positioncorresponding to amino acid 573 of SEQ ID NO: 1.

The present disclosure provides a serum albumin conjugate comprising theserum albumin variant, or functional fragment thereof, according to thepresent disclosure, and a compound.

In one example, a serum albumin conjugate of the present disclosure hasa longer serum half-life compared to a serum albumin conjugatecomprising a serum albumin set forth in SEQ ID NO: 1. Examples ofincreased serum half-life and assays for determining serum half-life aredescribed herein and are to be taken to apply mutatis mutandis to thisexample of the disclosure.

In another example, a serum albumin conjugate of the present disclosurehas an increased binding affinity for FcRn compared to a serum albuminconjugate comprising a serum albumin set forth in SEQ ID NO: 1. Forexample, the level of binding of the serum albumin conjugate to FcRn isincreased by at least about 2 fold or 4 fold or 5 fold or 10 fold. Forexample, the level of binding to the FcRn is increased by at least about2 fold or 10 fold or 40 fold or 100 fold or 150 fold. Examples ofincreased binding affinity for FcRn and assays for determining same aredescribed herein and are to be taken to apply mutatis mutandis to thisexample of the disclosure.

In one example, the serum albumin variant, or functional fragmentthereof, is conjugated to another compound and/or encapsulates anothercompound. Compounds contemplated by the present disclosure can take anyof a variety of forms including natural compounds, chemical smallmolecule compounds or biological compounds.

In one example, the serum albumin variant, or functional fragmentthereof, of the present disclosure is conjugated to a compound, which isdirectly or indirectly bound to the serum albumin variant, or functionalfragment thereof.

Exemplary compounds include a protein, a protein comprising an antibodyvariable region, an antibody mimetic, a domain antibody, a toxin, aradioisotope, a detectable label, a peptide, a polypeptide, a colloid, achemotherapeutic agent, a nucleic acid, a small molecule, antisenseoligonucleotide, a short hairpin RNA (shRNA), a siRNA, an interferingRNA (RNAi), a ribozyme, a microRNA, a microRNA adapted shRNA (shRNAmir),a DNAzyme and mixtures thereof. In one example, the detectable label isan imaging agent.

In one example, the compound is a protein-based compound, e.g., apeptide, polypeptide or protein. In one example, the protein is atherapeutic protein.

In another example, the compound is a protein (e.g., a therapeuticprotein) comprising a non-antibody antigen binding domain, such as anadnectin, an affibody, an atrimer, an evasin, a designed ankyrin-repeatprotein (DARPin) or an anticalin.

In one example, the compound is a protein (e.g., a therapeutic protein)comprising a variable region fragment (Fv). For example, the protein isselected from the group consisting of:

-   -   (i) a single chain Fv fragment (scFv);    -   (ii) a dimeric scFv (di-scFv); or    -   (iii) a diabody;    -   (iv) a triabody;    -   (v) a tetrabody;    -   (vi) a Fab;    -   (vii) a F(ab′)₂;    -   (viii) a Fv; or    -   (ix) one of (i) to (viii) linked to a constant region of an        antibody, Fc or a heavy chain constant domain (C_(H)) 2 and/or        C_(H)3.

In one example, the protein is an antibody or antigen binding fragment.

In one example, an antibody or antigen binding fragment of the presentdisclosure is recombinant, chimeric, CDR grafted, humanized,synhumanized, primatized, deimmunized or human.

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a von Willebrand factor,or modified form thereof. For example, the compound is a von Willebrandfactor, or a modified form thereof. In one example, the von Willebrandfactor comprises a D′D3 domain.

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a complement inhibitor ormodified form thereof. For example, the compound is a complementinhibitor, or a modified form thereof. In one example, the complementinhibitor is selected from the group consisting of Factor I, (fI),Factor H (fH), C4b-binding protein (C4 bp), soluble CD55 (decayaccelerating factor (DAF)), C1-inhibitor (C1-INH or C1 esteraseinhibitor); soluble CD35 (sCR1); soluble CD46 (membrane cofactor protein(MCP)), soluble CD59 (protectin), TT30 (CR2-fH), Cobra venom factor(CVF) and a functional fragment or variant thereof.

In one example, the complement inhibitor is a soluble complementinhibitor, such as sCR1, or a functional fragment or variant thereof. Inone example, the complement inhibitor is a variant or modified sCR1.

In one example, the complement inhibitor is a C1-inhibitor (i.e.,C1-INH), or a functional fragment or variant thereof. In one example,the complement inhibitor is a variant of modified C1-INH.

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a blood coagulationfactor.

In another example, the present disclosure provides a serum albuminvariant, or functional fragment thereof, conjugated to a compound thatbinds a blood coagulation factor. For example, the compound is or bindsto a blood coagulation factor.

In one example, the blood coagulation factor is selected from the groupconsisting of Factor I, Factor II (prothrombin)/thrombin, Factor III,Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI,Factor XII Factor XIII and an activated form of any of the foregoing.For example, the blood coagulation factor is Factor IX and/or FactorIXa. In another example, the blood coagulation factor is Factor X and/orFactor Xa. In a further example, the blood coagulation factor is FactorIX/IXa and Factor X/Xa. In one example, the blood coagulation factor isFactor VII and/or Factor VIIa. In another example, the blood coagulationfactor is Factor VIII and/or Factor Vila.

The present disclosure also provides a composition comprising a serumalbumin conjugate of the disclosure and a pharmaceutical carrier and/orexcipient.

In one example, the composition has increased binding affinity for FcRncompared to a composition comprising a serum albumin conjugatecomprising a serum albumin comprising a sequence set forth in SEQ ID NO:1.

In one example, the composition has increased serum half-life comparedto a composition comprising a serum albumin conjugate comprising a serumalbumin comprising a sequence set forth in SEQ ID NO: 1.

The present disclosure also provides a method of treating or preventinga disease or condition in a subject, the method comprising administeringthe serum albumin conjugate of the present disclosure, or thecomposition comprising the serum albumin variant, or functional fragmentthereof. In one example, the subject is in need thereof.

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, or a serum albumin conjugate, or acomposition comprising the serum albumin variant or functional fragmentthereof, for use in treating or preventing a disease or condition in asubject.

In one example, the present disclosure provides use of the serum albuminconjugate of the present disclosure, or the composition comprising theserum albumin variant, or functional fragment thereof, of the presentdisclosure, in the manufacture of a medicament for the treatment orprevention of a disease or condition in a subject.

In one example, the disease or condition is a bleeding disorder.

In one example, the subject suffers from a bleeding disorder. In oneexample, the subject has been diagnosed as suffering from a bleedingdisorder. In one example, the subject is receiving treatment for ableeding disorder.

In one example of any method described herein, the serum albuminconjugate or composition comprising the serum albumin variant of thepresent disclosure is administered before or after the development of ableeding disorder. In one example of any method described herein, theserum albumin conjugate or composition comprising the serum albuminvariant of the present disclosure is administered before the developmentof the bleeding disorder. In one example of any method described herein,the serum albumin conjugate or composition comprising the serum albuminvariant of the present disclosure is administered after the developmentof the bleeding disorder.

In one example of any method described herein, the serum albuminconjugate or composition comprising the serum albumin variant of thepresent disclosure is administered before or after the onset of ableeding event. In one example, the serum albumin conjugate orcomposition comprising the serum albumin variant of the presentdisclosure is administered before the onset of a bleeding event. Inanother example, the serum albumin conjugate or composition comprisingthe serum albumin variant of the present disclosure is administeredafter the onset of a bleeding event.

A bleeding event will be apparent to the skilled person and include, forexample a minor and/or major bleeding event. In one example, thebleeding event is a major bleeding event. For example, a major bleedingevent is any episode of bleeding that leads to ≥5 g/dL reducedhaemoglobin or a ≥15% absolute decrease in haematocrit. In one example,the bleeding event is a minor bleeding event. For example, a minorbleeding event is any episode of bleeding that leads to ≤4 g/dL reducedhaemoglobin or a ≥10% absolute decrease in haematocrit.

In one example, the subject is at risk of developing a bleedingdisorder. For example, a subject at risk of developing a bleedingdisorder includes, but is not limited, to those with a mutation,deletion or rearrangement in a blood coagulation factor, e.g., FactorVII and/or Factor IX, or those with a platelet disorder. In one example,the subject has a relative that has developed a bleeding disorder. Forexample, the bleeding disorder is inherited. In one example, thebleeding disorder is acquired.

In one example, the serum albumin conjugate or composition comprisingthe serum albumin variant is administered before or after the onset ofsymptoms of a bleeding disorder. In one example, the serum albuminconjugate or composition comprising the serum albumin variant isadministered before the onset of symptoms of a bleeding disorder. In oneexample, the serum albumin conjugate or composition comprising the serumalbumin variant is administered after the onset of symptoms of ableeding disorder. In one example, the serum albumin conjugate orcomposition comprising the serum albumin variant of the presentdisclosure is administered at a dose that alleviates or reduces one ormore of the symptoms of a bleeding disorder.

Symptoms of a bleeding disorder will be apparent to the skilled personand include, for example:

Easy bruising;

Bleeding gums;

Heavy bleeding from small cuts or dental work;

Unexplained nosebleeds;

Heavy menstrual bleeding;

Bleeding into joints; and/or

Excessive bleeding following surgery.

In one example, the bleeding disorder is caused by a blood coagulationdisorder. For example, the blood coagulation disorder is haemophilia,von Willebrand disease, Factor I deficiency, Factor II deficiency,Factor V deficiency, combined Factor V/Factor VIII deficiency, FactorVII deficiency, Factor X deficiency, Factor XI deficiency or Factor XIIIdeficiency. In one example, the haemophilia is haemophilia A orhaemophilia B. In one example, the subject has a condition requiringprophylactic treatment.

In one example, the serum albumin conjugate or composition comprisingthe serum albumin variant of the present disclosure is administered tothe subject in an amount to reduce the severity of the bleeding in thesubject.

In one example of any method described herein, the subject is a mammal,for example a primate such as a human.

Methods of treatment described herein can additionally compriseadministering a further compound to reduce, treat or prevent the effectof the bleeding disorder.

The present disclosure also provides a composition comprising a serumalbumin variant, or functional fragment thereof, that binds to a bloodcoagulation factor for use in treating or preventing a bleedingdisorder.

The present disclosure also provides use of a composition comprising aserum albumin variant, or functional fragment thereof, that binds to ablood coagulation factor in the manufacture of a medicament for treatingor preventing a bleeding disorder.

The present disclosure also provides a kit comprising at least one serumalbumin conjugate or composition comprising a serum albumin variant, orfunctional fragment thereof of the disclosure that binds to a bloodcoagulation factor packaged with instructions for use in treating orpreventing a bleeding disorder in a subject. Optionally, the kitadditionally comprises a therapeutically active compound or drug.

The present disclosure also provides a kit comprising at least one serumalbumin conjugate or composition comprising a serum albumin variant, orfunctional fragment thereof of the disclosure that binds to a bloodcoagulation factor packaged with instructions to administer theconjugate or composition to a subject who is suffering from or at riskof suffering from a bleeding disorder, optionally, in combination with atherapeutically active compound or drug.

Exemplary effects of serum albumin conjugates or compositions of thepresent disclosure that bind to a blood coagulation factor are describedherein and are to be taken to apply mutatis mutandis to the examples ofthe disclosure set out in the previous five paragraphs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graphical representation showing the serum HSA concentrationof HSA variants injected into mice expressing human FcRn receptor.(μg/mL; mean±SEM).

KEY TO SEQUENCE LISTING

SEQ ID NO: 1 amino acid sequence of human serum albumin

SEQ ID NO: 2 amino acid sequence of human coagulation Factor VIII

SEQ ID NO: 3 amino acid sequence of human coagulation Factor IX

SEQ ID NO: 4 amino acid sequence of human coagulation Factor X

SEQ ID NO: 5 amino acid sequence of human coagulation Factor VII

SEQ ID NO: 6 amino acid sequence of human von Willebrand factor

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or groups of compositionsof matter.

Those skilled in the art will appreciate that the present disclosure issusceptible to variations and modifications other than thosespecifically described. It is to be understood that the disclosureincludes all such variations and modifications. The disclosure alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specificexamples described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to applymutatis mutandis to any other example of the disclosure unlessspecifically stated otherwise. Stated another way, any specific exampleof the present disclosure may be combined with any other specificexample of the disclosure (except where mutually exclusive).

Any example of the present disclosure disclosing a specific feature orgroup of features or method or method steps will be taken to provideexplicit support for disclaiming the specific feature or group offeatures or method or method steps.

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (for example, in cellculture, molecular genetics, immunology, immunohistochemistry, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present disclosure are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The description and definitions of variable regions and parts thereof,immunoglobulins, antibodies and fragments thereof herein may be furtherclarified by the discussion in Kabat Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,1987 and 1991, Bork et al., J Mol. Biol. 242, 309-320, 1994, Chothia andLesk J. Mol Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883,1989 and/or or Al-Lazikani et al., J Mol Biol 273, 927-948, 1997.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source.

Selected Definitions

Serum albumin, or blood albumin, is the most abundant blood protein andfunctions as a carrier protein for steroids, fatty acids and thyroidhormones in the blood, as well as playing a major role in stabilisingextracellular fluid volume. For the purposes of nomenclature only andnot limitation an exemplary sequence of a human serum albumin is set outin NCBI GenBank Accession ID: AEE60908 and SEQ ID NO: 1. It should beunderstood that reference to “serum albumin” or “albumin” includespreproalbumin, which comprises the N-terminal peptide, proalbumin andthe secreted albumin. Positions of amino acids are referred to herein byreference to the secreted albumin protein consisting of 585 amino acids(e.g., as set out in SEQ ID NO: 1). Albumin comprises three homologousdomains, wherein each domain is a product of two subdomains that possesscommon structural motifs. Domains I, II and III may be defined withreference to human serum albumin (as set forth in SEQ ID NO: 1). Forexample, domain I comprises amino acids 1 (±1 to 15 amino acids) to 194(±1 to 15 amino acids) of SEQ ID NO: 1, domain II comprises amino acids192 (±1 to 15 amino acids) to 387 (±1 to 15 amino acids) of SEQ ID NO: 1and domain III comprises amino acid residues 381 (±1 to 15 amino acids)to 585 (±1 to 15 amino acids) of SEQ ID NO: 1. The phrase “±1 to 15amino acids” means that the amino acid residue may deviate by 1 or 2 or3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15amino acids to the C-terminus and/or the N-terminus of the stated aminoacid position. Exemplary domains I, II and III are described by Dockalet al (The Journal of Biological Chemistry, 1999, Vol. 274(41):29303-29310) and Kjeldsen et al (Protein Expression and Purification,1998, Vol 13: 163-169).

Additional sequences of serum albumin from other species (e.g., primateserum albumin, (such as chimpanzee serum albumin, gorilla serumalbumin), rodent serum albumin (such as hamster serum albumin, guineapig serum albumin, mouse albumin and rat serum albumin), bovine serumalbumin, equine serum albumin, donkey serum albumin, rabbit serumalbumin, goat serum albumin, sheep serum albumin, dog serum albumin,chicken serum albumin and pig serum albumin) can be determined usingsequences provided herein and/or in publically available databasesand/or determined using standard techniques (e.g., as described inAusubel et al., (editors), Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience (1988, including allupdates until present) or Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

As used herein the phrase “corresponding to” in reference to theposition of an amino acid in SEQ ID NO: 1 should be understood asreference to an amino acid residue or position within an albuminsequence, and not necessarily a sequence comprising SEQ ID NO: 1. Forexample, reference to “a position corresponding to amino acid 522 of SEQID NO: 1” in an albumin sequence comprising a 10 amino acid N-terminaltruncation would necessarily refer to an amino acid at position 512. Inone example, the serum albumin comprises a sequence set forth in SEQ IDNO: 1.

Reference to a “functional fragment” of serum albumin should beunderstood as a reference to a fragment of serum albumin which haveretained and exhibit serum albumin functionality (i.e., ability to bindto FcRn). A fragment may comprise or consist of one more domains ofalbumin, fragments of such domains or combinations thereof.

As used herein “amino acid substitution(s)” refers to the replacement ofan amino acid at a particular position in a polypeptide sequence withanother amino acid.

As used herein, the term “FcRn” refers to the neonatal Fc receptor, alsoknown as the Brambell receptor, and is a heterodimer of truncated heavychain of the major histocompatibility complex class 1-like Fc receptor(FCGRT) and beta-2-microglobulin.

As used herein, the terms “variant” or “mutant” or “mutated” refers to aserum albumin which has undergone substitution of one or more aminoacids using well known techniques for site directed mutagenesis or anyother conventional method.

As used herein, the term “binds” in reference to the interaction of aserum albumin with FcRn means that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on a cell or protein.

As used herein, phrases referring to “increasing affinity” or “increasedbinding” or “binding being at a higher level” in relation to theinteraction of a serum albumin variant with FcRn will be understood tomean that a serum albumin variant, or functional fragment thereof, bindsto or associates with FcRn more frequently, more rapidly, with greaterduration and/or greater affinity than a human serum albumin as set forthin SEQ ID NO: 1 (e.g., 1.5 fold or 2 fold or 5 fold or 10 fold or 20fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200fold).

For the purposes of clarification and as will be apparent to the skilledartisan based on the exemplified subject matter herein, reference to“affinity” in this specification is a reference to the interaction,binding or association of a serum albumin variant with FcRn.

For the purposes of clarification and as will be apparent to the skilledartisan based on the description herein, reference to an “affinity of atleast about” will be understood to mean that the affinity is equal tothe recited value or higher (i.e., the value recited as the affinity islower), i.e., an affinity of 2 nM is greater than an affinity of 3 nM.Stated another way, this term could be “an affinity of X or less”,wherein X is a value recited herein.

As used herein, the term “serum half-life” or “plasma half-life” in thecontext of the present disclosure refers to the period of time requiredfor the concentration or amount of serum albumin in the body to bereduced by 50% (i.e., one half) for example due to degradation and/orclearance or sequestration by natural mechanisms. The skilled personwould recognise that the serum half-life of serum albumin in a subjectis dependent on various physiological conditions (e.g., health status,body size/weight). In a healthy human subject, the serum half-life ofserum albumin is 19 days. Methods for determining the serum half-life ofserum albumin are known in the art and include, for example,pharmacokinetic analysis. For the purposes of the present disclosure, an“increase” or “enhanced” serum half-life refers to an elevation orincrease in time taken for the serum concentration of the serum albuminvariant to be reduced by 50%, compared to a serum albumin set forth inSEQ ID NO: 1.

The term “recombinant” shall be understood to mean the product ofartificial genetic recombination. Accordingly, in the context of arecombinant protein comprising an antibody antigen binding domain, thisterm does not encompass an antibody naturally-occurring within asubject's body that is the product of natural recombination that occursduring B cell maturation. However, if such an antibody is isolated, itis to be considered an isolated protein comprising an antibody antigenbinding domain. Similarly, if nucleic acid encoding the protein isisolated and expressed using recombinant means, the resulting protein isa recombinant protein comprising an antibody antigen binding domain. Arecombinant protein also encompasses a protein expressed by artificialrecombinant means when it is within a cell, tissue or subject, e.g., inwhich it is expressed.

The term “protein” shall be taken to include a single polypeptide chain,i.e., a series of contiguous amino acids linked by peptide bonds or aseries of polypeptide chains covalently or non-covalently linked to oneanother (i.e., a polypeptide complex). For example, the series ofpolypeptide chains can be covalently linked using a suitable chemical ora disulfide bond. Examples of non-covalent bonds include hydrogen bonds,ionic bonds, Van der Waals forces, and hydrophobic interactions.

The term “polypeptide” or “polypeptide chain” will be understood fromthe foregoing paragraph to mean a series of contiguous amino acidslinked by peptide bonds.

As used herein, the term “antigen binding domain” shall be taken to meana region of an antibody that is capable of specifically binding to anantigen, i.e., a V_(H) or a V_(L) or an Fv comprising both a V_(H) and aV_(L). The antigen binding domain need not be in the context of anentire antibody, e.g., it can be in isolation (e.g., a domain antibody)or in another form, e.g., as described herein, such as a scFv.

For the purposes for the present disclosure, the term “antibody”includes a protein capable of specifically binding to one or a fewclosely related antigens (e.g., a blood coagulation factor) by virtue ofan antigen binding domain contained within a Fv. This term includes fourchain antibodies (e.g., two light chains and two heavy chains),recombinant or modified antibodies (e.g., chimeric antibodies, humanizedantibodies, human antibodies, CDR-grafted antibodies, primatizedantibodies, de-immunized antibodies, synhumanized antibodies,half-antibodies, bispecific antibodies). Antibodies can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁ and IgA₂) or subclass. In one example, the antibody is amurine (mouse or rat) antibody or a primate (such as, human) antibody.In one example the antibody heavy chain is missing a C-terminal lysineresidue. In one example, the antibody is humanized, synhumanized,chimeric, CDR-grafted or deimmunized.

As used herein, “variable region” refers to the portions of the lightand/or heavy chains of an antibody as defined herein that is capable ofspecifically binding to an antigen and, includes amino acid sequences ofcomplementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3,and framework regions (FRs).

As used herein, the term “Fv” shall be taken to mean any protein,whether comprised of multiple polypeptides or a single polypeptide, inwhich a variable region of the light chain (V_(L)) and a variable regionof a heavy chain (V_(H)) associate and form a complex having an antigenbinding domain, i.e., capable of specifically binding to an antigen. TheV_(H) and the V_(L) which form the antigen binding domain can be in asingle polypeptide chain or in different polypeptide chains.Furthermore, an Fv of the disclosure (as well as any protein of thedisclosure) may have multiple antigen binding domains which may or maynot bind the same antigen. This term shall be understood to encompassfragments directly derived from an antibody as well as proteinscorresponding to such a fragment produced using recombinant means. Insome examples, the V_(H) is not linked to a heavy chain constant domain(C_(H)) 1 and/or the V_(L) is not linked to a light chain constantdomain (C_(L)). Exemplary Fv containing polypeptides or proteins includea Fab fragment, a Fab′ fragment, a F(ab′) fragment, a scFv, a diabody, atriabody, a tetrabody or higher order complex, or any of the foregoinglinked to a constant region or domain thereof, e.g., C_(H)2 or C_(H)3domain, e.g., a minibody. A “Fab fragment” consists of a monovalentantigen-binding fragment of an immunoglobulin, and can be produced bydigestion of a whole antibody with the enzyme papain, to yield afragment consisting of an intact light chain and a portion of a heavychain or can be produced using recombinant means. A “Fab′ fragment” ofan antibody can be obtained by treating a whole antibody with pepsin,followed by reduction, to yield a molecule consisting of an intact lightchain and a portion of a heavy chain comprising a V_(H) and a singleconstant domain. Two Fab′ fragments are obtained per antibody treated inthis manner A Fab′ fragment can also be produced by recombinant means. A“F(ab′)2 fragment” of an antibody consists of a dimer of two Fab′fragments held together by two disulfide bonds, and is obtained bytreating a whole antibody molecule with the enzyme pepsin, withoutsubsequent reduction. A “Fab₂” fragment is a recombinant fragmentcomprising two Fab fragments linked using, for example a leucine zipperor a C_(H)3 domain. A “single chain Fv” or “scFv” is a recombinantmolecule containing the variable region fragment (Fv) of an antibody inwhich the variable region of the light chain and the variable region ofthe heavy chain are covalently linked by a suitable, flexiblepolypeptide linker.

An “antigen binding fragment” of an antibody comprises one or morevariable regions of an intact antibody. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)₂ and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules, half antibodies andmultispecific antibodies formed from antibody fragments.

As used herein, the term “condition” refers to a disruption of orinterference with normal function, and is not to be limited to anyspecific condition, and will include diseases or disorders.

As used herein, the term “bleeding condition” or “bleeding disorder”refers to a condition in which there is abnormal blood coagulation,e.g., reduced or insufficient blood coagulation capability and/orabnormal bleeding (internal and/or external), e.g., excessive bleeding.

As used herein, “coagulation factor” refers to a factor that isassociated with the formation of a blot clot, i.e., blood coagulation.In one example, the coagulation factor has pro-coagulant activity.Coagulation factors are known in the art and include without limitationFactor I, Factor II, Factor III, Factor V, Factor VII, Factor VIII,Factor IX, Factor X, Factor XI, Factor XII and Factor XIII or anactivated form of any of the foregoing. This term also includesrecombinant forms of coagulation factors and/or modified forms thereof,e.g., as is known in the art and/or described herein.

As used herein, a subject “at risk” of developing a disease or conditionor relapse thereof or relapsing may or may not have detectable diseaseor symptoms of disease, and may or may not have displayed detectabledisease or symptoms of disease prior to the treatment according to thepresent disclosure. “At risk” denotes that a subject has one or morerisk factors, which are measurable parameters that correlate withdevelopment of the disease or condition, as known in the art and/ordescribed herein.

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a serum albumin variant conjugate described herein tothereby reduce or eliminate at least one symptom of a specified diseaseor condition or to slow progression of the disease or condition.

As used herein, the term “preventing”, “prevent” or “prevention”includes providing prophylaxis with respect to occurrence or recurrenceof a bleeding disease or a symptom of a bleeding disease in anindividual. An individual may be predisposed to or at risk of developingthe disease or disease relapse but has not yet been diagnosed with thedisease or the relapse.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired result. Forexample, the desired result may be a therapeutic or prophylactic result.An effective amount can be provided in one or more administrations. Insome examples of the present disclosure, the term “effective amount” ismeant an amount necessary to effect treatment of a disease or conditionas hereinbefore described. In some examples of the present disclosure,the term “effective amount” is meant an amount necessary to effect achange in a factor associated with a disease or condition ashereinbefore described. For example, the effective amount may besufficient to effect a change in the level of coagulation. The effectiveamount may vary according to the disease or condition to be treated orfactor to be altered and also according to the weight, age, racialbackground, sex, health and/or physical condition and other factorsrelevant to the mammal being treated. Typically, the effective amountwill fall within a relatively broad range (e.g., a “dosage” range) thatcan be determined through routine trial and experimentation by a medicalpractitioner. Accordingly, this term is not to be construed to limit thedisclosure to a specific quantity. The effective amount can beadministered in a single dose or in a dose repeated once or severaltimes over a treatment period.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disease or condition. A therapeutically effective amountherein may vary according to factors such as the disease state, age,sex, and weight of the patient, and the ability of the albumin conjugateto elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the albumin conjugate are outweighed by the therapeuticallybeneficial effects. In one example, a therapeutically effective amountshall be taken to mean a sufficient quantity of albumin conjugate toreduce or inhibit one or more symptoms of a bleeding disorder or acomplication thereof.

As used herein, the term “prophylactically effective amount” shall betaken to mean a sufficient quantity of the albumin conjugate to preventor inhibit or delay the onset of one or more detectable symptoms of ableeding disorder or a complication thereof.

As used herein, the term “subject” shall be taken to mean any animalincluding humans, for example a mammal. Exemplary subjects include butare not limited to humans and non-human primates. For example, thesubject is a human.

Serum Albumin Variants

The present disclosure provides serum albumin variants, or functionalfragments thereof, with defined amino acid substitutions compared to asequence set forth in SEQ ID NO: 1. In one example, a serum albuminvariant, or fragment thereof, of the present disclosure comprises asequence at least about 85% or 90% or 95% or 97% or 98% or 99% identicalto a sequence disclosed herein, wherein the serum albumin variant, orfragment thereof, binds to FcRn as described herein according to anyexample.

The present disclosure provides a serum albumin variant, or functionalfragment thereof, comprising one or more amino acid substitutionsselected from the group consisting of:

-   -   (i) an amino acid selected from the group consisting of glycine,        isoleucine, lysine, methionine, phenylalanine, tryptophan,        tyrosine, valine and leucine substituted for glutamine at a        position corresponding to amino acid 522 of SEQ ID NO: 1;    -   (ii) valine substituted for alanine at a position corresponding        to amino acid 552 of SEQ ID NO: 1;    -   (iii) an amino acid selected from the group consisting of        alanine, glutamic acid, histidine, serine, lysine and arginine        substituted for glycine at a position corresponding to amino        acid 572 of SEQ ID NO: 1; and    -   (iv) combinations thereof.

In one example, the serum albumin variant, or functional fragmentthereof further comprises tyrosine substituted for lysine at a positioncorresponding to amino acid 573 of SEQ ID NO: 1.

For example, the inventors have identified several amino acid residuesin a sequence set forth in SEQ ID NO: 1 that can be substituted withoutloss of function or that result in improved function. In one example,the serum albumin variant, or functional fragment thereof, comprisesbetween 1 and 3 amino acid substitutions at a position corresponding toamino acid 522, 552 and/or 572 compared to a sequence set forth in SEQID NO: 1. For example, the serum albumin variant, or functional fragmentthereof, comprises 1 or 2 or 3 amino acid substitutions at a positioncorresponding to amino acid 522, 552 and/or 572 compared to a sequenceset forth in SEQ ID NO: 1

Optionally, the serum albumin variant, or functional fragment thereofadditionally comprises 1 amino acid substitution at a positioncorresponding to amino acid 573 compared to a sequence set forth in SEQID NO: 1. In one example, the serum albumin variant, or functionalfragment thereof, comprises between 1 and 4 amino acid substitutions ata position corresponding to amino acid 522, 552 and/or 572 andoptionally 573 compared to a sequence set forth in SEQ ID NO: 1. Forexample, the serum albumin variant or functional fragment thereof,comprises 1 or 2 or 3 or 4 amino acid substitutions at a positioncorresponding to amino acid 522, 552 and/or 572 and optionally 573compared to a sequence set forth in SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a glycineat a position corresponding to amino acid 522 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises aisoleucine at a position corresponding to amino acid 522 of SEQ ID NO:1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a lysineat a position corresponding to amino acid 522 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises amethionine at a position corresponding to amino acid 522 of SEQ ID NO:1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises aphenylalanine at a position corresponding to amino acid 522 of SEQ IDNO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises atryptophan at a position corresponding to amino acid 522 of SEQ ID NO:1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a tyrosineat a position corresponding to amino acid 522 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a valineat a position corresponding to amino acid 522 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises an alanineat a position corresponding to amino acid 522 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a valineat a position corresponding to amino acid 552 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises an alanineat a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a glutamicacid at a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises ahistidine at a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a serineat a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a lysineat a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises anarginine at a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a valineat a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1 and anarginine at a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1 and avaline at a position corresponding to amino acid 552 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1 and atyrosine at a position corresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a valineat a position corresponding to amino acid 552 of SEQ ID NO: 1 and atyrosine at a position corresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises anarginine at a position corresponding to amino acid 572 of SEQ ID NO: 1and a tyrosine at a position corresponding to amino acid 573 of SEQ IDNO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1, a valineat a position corresponding to amino acid 552 of SEQ ID NO: 1, and anarginine at a position corresponding to amino acid 572 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1, a valineat a position corresponding to amino acid 552 of SEQ ID NO: 1, and atyrosine at a position corresponding to amino acid 573 of SEQ ID NO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1, anarginine at a position corresponding to amino acid 572 of SEQ ID NO: 1,and a tyrosine at a position corresponding to amino acid 573 of SEQ IDNO: 1.

In one example, a serum albumin variant, or functional fragment thereof,of the present disclosure comprises a variant of a sequence set forth inSEQ ID NO: 1, wherein the variant sequence at least comprises a leucineat a position corresponding to amino acid 522 of SEQ ID NO: 1, a valineat a position corresponding to amino acid 552 of SEQ ID NO: 1, anarginine at a position corresponding to amino acid 572 of SEQ ID NO: 1,and a tyrosine at a position corresponding to amino acid 573 of SEQ IDNO: 1.

Exemplary methods for producing variant forms of serum albumin aredescribed herein or known in the art and include:

-   -   mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525:        309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett.        107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7: 18,        2007; and WO1999/058661);    -   introducing a nucleic acid encoding the polypeptide into a        mutator cell, e.g., XL-1Red, XL-mutS and XL-mutS-Kanr bacterial        cells (Stratagene);    -   DNA shuffling, e.g., as disclosed in Stemmer, Nature 370:        389-91, 1994; and    -   site directed mutagenesis, e.g., as described in Dieffenbach        (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual,        Cold Spring Harbor Laboratories, N Y, 1995).

Exemplary methods for determining biological activity of the serumalbumin variant, or functional fragment thereof, of the disclosure willbe apparent to the skilled person and/or described herein, e.g., FcRnaffinity. For example, methods for determining affinity of the serumalbumin variant, or functional fragment thereof, include affinity,association, dissociation and therapeutic efficacy are described herein.

For example, the inventors have identified several amino acid residuesin a sequence set forth in SEQ ID NO: 1 that can be substituted toincrease the half-life of the serum albumin. For example, the serumalbumin variant, or functional fragment thereof comprises one or moreamino acid substitutions that increase the affinity of the albumin forthe neonatal Fc receptor (FcRn). In one example, the variant orfunctional fragment thereof binds with increased affinity to FcRncompared to a serum albumin set forth in SEQ ID NO: 1. For example, theserum albumin variant, or functional fragment thereof, has increasedaffinity for FcRn at lower pH, e.g., about pH 6.0, to facilitatealbumin/FcRn binding in an endosome. In one example, the albumin hasincreased affinity for FcRn at about pH 6 compared to its affinity atabout pH 7.4, which facilitates the re-release of albumin into bloodfollowing cellular recycling. These amino acid substitutions are usefulfor extending the half-life of a protein, by reducing clearance from theblood.

In one example, exemplary amino acid substitutions include Q522G, Q522I,Q522K, Q522M, Q522L, Q522F, Q522W, Q522Y, Q522V, A552V, G572A, G572E,G572H, G572S, G572K and G572R.

In another example, exemplary amino acid substitutions include Q522G,Q522I, Q522K, Q522M, Q522L, Q522F, Q522W, Q522Y, Q522V, Q522A, A552V,G572A, G572E, G572H, G572S, G572K, G572V and G572R

Conjugates

In one example, a serum albumin variant or functional fragment thereof,of the present disclosure is conjugated to a compound and/orencapsulates another compound. For example, the compound is selectedfrom the group consisting of a radioisotope, a detectable label, atherapeutic compound, a therapeutic protein, an imaging agent, acolloid, a toxin, a nucleic acid, a peptide, a protein, a smallmolecule, antisense oligonucleotide, a short hairpin RNA (shRNA), asiRNA, an interfering RNA (RNAi), a ribozyme, a microRNA, a microRNAadapted shRNA (shRNAmir), a DNAzyme and mixtures thereof.

The compound can be directly or indirectly bound to the serum albuminvariant or functional fragment thereof (e.g., can comprise a linker inthe case of indirect binding). Examples of compounds include, aradioisotope (e.g., iodine-131, yttrium-90 or indium-111), a detectablelabel (e.g., a fluorophore or a fluorescent nanocrystal or quantum dot),a therapeutic compound or protein (e.g., a chemotherapeutic or ananti-inflammatory or coagulation factor), a colloid (e.g., gold), atoxin (e.g., ricin or tetanus toxoid), a nucleic acid, a protein (e.g.,a protein comprising an antigen binding domain of an antibody), andmixtures thereof. In one example, the serum albumin variant orfunctional fragment thereof, is conjugated to a coagulation factor.

For example, the compound is a protein and is conjugated to the serumalbumin variant or functional fragment thereof by an amine bond.

In one example, disclosure provides a fusion protein comprising theserum albumin variant or functional fragment thereof and the compound(e.g., a therapeutic protein, such as a coagulation factor). Forexample, the compound is positioned at N-terminus of the serum albuminvariant or functional fragment thereof, C-terminus of the serum albuminvariant or functional fragment thereof, inserted into a loop in theserum albumin variant or functional fragment thereof or any combinationthereof.

Exemplary compounds that can be conjugated to a serum albumin variant ofthe disclosure and methods for such conjugation are known in the art anddescribed herein.

Radioisotopes

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a radioisotope.

Radioisotopes suitable for use in the present disclosure will beapparent to the skilled person and include, for example, iodine-123(¹²³I), iodine-125 (¹²⁵I), iodine-130 (¹³⁰I), iodine-133 (¹³³I),iodine-135 (¹³⁵I), scandium-47 (⁴⁷Sc), arsenic-72 (⁷²As), scandium-72(⁷²Sc), yttrium-90 (⁹⁰Y), yttrium-88 (⁸⁸Y), ruthenium-97 (⁹⁷Ru),palladium-100 (¹⁰⁰Pd) rhodium-101m (^(101m)Rh), antimony 119 (¹¹⁹Sb),barium 128 (¹²⁸Ba), mercury-197 (¹⁹⁷Hg), astatine-211 (²¹¹At),bismuth-212 (²¹²Bi) samarium 153 (¹⁵³Sm), europium 169 (¹⁶⁹Eu), lead 212(212Pb), palladium-109 (¹⁹⁹Pd), indium-111 (¹¹¹In), ⁶⁷Gu, ⁶⁸Gu,Copper-67 (⁶⁷Cu), bromine-75 (⁷⁵Br), bromine-76 (⁷⁶Br), bromine-77(⁷⁷Br), technetium-99m (^(99m)Tc), carbon-11 (¹¹C), nitrogen-13 (¹³N),oxygen-15 (¹⁵O), iodine-18 (¹⁸4 rhenium-188 (¹⁸⁸Re), lead-203 (²⁰³Pb),copper-64 (⁶⁴Cu), rhodium-105 (¹⁰⁵Rh), gold-198 (¹⁹⁸Au), argon-199(¹⁹⁹Ag) or lutetium-177 (¹⁷⁷Lu).

Detectable Labels

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a detectable label. Forexample, the detectable label is a fluorophore, a fluorescentnanocrystal or quantum dot.

The term “fluorophore” refers to a chemical compound that absorbs lightat a specific wavelength and fluoresces, or re-emits light, at a longerwavelength. Fluorophores may fluoresce in the ultraviolet spectrum (10nm to 400 nm), visible spectrum (400 nm to 700 nm), or near infraredregion (680 nm to 100,000 nm).

Suitable fluorophores for use in the present disclosure will be apparentto the skilled and include, for example, indocyanine green, IRDye78,IRDye80, IRDye38, IRDye40, IRDye41, IRDye700, IRDye800, IRDye800CW, Cy5,Cy5.5, Cy7, DRAQSNO, Licor NIR, Alexa Fluor488, Alexa Fluor680, AlexaFluor 700, Alexa Fluor750, La Jolla Blue, R-phycoerythrin (PE),hydroxycoumarin, methoxycoumarin, aminocoumarin, Fluorescein FITC,Rhodamine Red-X, Texas Red, Allophycocyanin (APC) and analogs thereof.

In one example, the detectable label is a quantum dot. Quantum dots area semiconductor nanocrystal with size-dependent optical and electronicproperties. Exemplary materials suitable for use as quantum dots includeZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP, GaAs, GaSb, InP, InAs, Sb,AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge, and Si and ternary and quaternarymixtures thereof.

In one example, the detectable label is a fluorescent protein. Suitablefluorescent proteins for use in the present disclosure will be apparentto the skilled person and include, for example, Renilla luciferase,green fluorescent protein (GFP), cyan fluorescent protein (CFP), redfluorescent protein (RFP), blue fluorescent protein (BFP), yellowfluorescent protein (YFB) and derivatives thereof.

Biological Entities

Coagulation Factors

The present disclosure provides a serum albumin variant, or functionalfragment thereof, conjugated to a coagulation factor.

Blood coagulation occurs through a cascade of stages involving releaseof several coagulation factors, ultimately resulting in the formation ofa blood clot containing insoluble fibrin. Exemplary coagulation factorsinclude, but are not limited to, Factor I (Fibrinogen), Factor II(Prothrombin/thrombin), Factor III (Tissue factor), Factor V (Labilefactor), Factor VII (Proconvertin), Factor VIII (Antihaemophilicfactor), Factor IX (Christmas factor), Factor X (Stuart-Prower factor),Factor XI (Plasma thromboplastin antecedent), Factor XII (Hageman(contact) factor) and Factor XIII (Fibrin-stabilizingfactor/Prekallikrein (Fletcher) factor/HMWK (Fitzgerald) factor).

For example, the compound is a coagulation factor and is conjugated tothe serum albumin variant or functional fragment thereof by an aminebond.

In one example, disclosure provides a fusion protein comprising theserum albumin variant or functional fragment thereof and a coagulationfactor. For example, the coagulation factor is positioned at N-terminusof the serum albumin variant or functional fragment thereof, C-terminusof the serum albumin variant or functional fragment thereof, insertedinto a loop in the serum albumin variant or functional fragment thereofor any combination thereof.

In one example, the coagulation factor is Factor VIII. For the purposesof nomenclature only and not limitation, exemplary sequences of humanFactor VIII are set out in NCBI Ref Seq ID NP_000123, protein accessionnumber NM_000132.3 and in SEQ ID NO: 2.

In one example, the coagulation factor is Factor IX. For the purposes ofnomenclature only and not limitation, exemplary sequences of humanFactor IX are set out in GenBank ID AAA98726.1 and in SEQ ID NO: 3.

In one example, the coagulation factor is Factor X. For the purposes ofnomenclature only and not limitation, exemplary sequences of humanFactor X are set out in Gene ID: 2159 and in SEQ ID NO: 4.

In one example, the coagulation factor is Factor VII. For the purposesof nomenclature only and not limitation, exemplary sequences of humanFactor VII are set out in Ref Seq ID NM_00131 and in SEQ ID NO: 5.

For the purposes of nomenclature only and not limitation, exemplarysequences of human Factor I are set out in NCBI Ref Seq ID NM_000508(alpha chain) and NM_005141 (beta chain), exemplary sequences of humanFactor II are set out in Ref Seq ID NM_000506, exemplary sequences ofhuman Factor III are set out in Ref Seq ID NM_001993, exemplarysequences of human Factor V are set out in Ref Seq ID NM_000130,exemplary sequences of human Factor XI are set out in Ref Seq IDNM_000128, exemplary sequences of human Factor XII are set out in RefSeq ID NM_000505, exemplary sequences of human Factor XIII are set outin Ref Seq ID NM_000129 (A chain) and NM_001994 (B chain).

Additional sequence of coagulation factors can be determined usingsequences provided herein and/or in publically available databasesand/or determined using standard techniques (e.g., as described inAusubel et al., (editors), Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience (1988, including allupdates until present) or Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

Exemplary coagulation factors may be plasma derived from a donor or arecombinant protein. For example, the coagulation factor is a plasmaderived or recombinant coagulation factor protein. For example, thetherapeutic protein is selected from the group consisting of Factor I(Fibrinogen), Factor II ((prothrombin)/thrombin), Factor III (TissueFactor), Factor V (Labile Factor), Factor VII (Proconvertin), FactorVIIa (e.g., NOVOSEVEN®), Factor VIII (Antihaemophilic Facto; such as asingle chain recombinant Factor VIII, e.g., as described in Zollner etal., Thromb Res. 132:280-287, 2013; or a plasma derived Factor VIIIproduct, such as FEIBA®, MONOCLATE-P®, or BIOSTATE®; or a recombinantFactor VIII product, such as ADVATE®, ELOCTATE®, RECOMBINATE®, KOGENATEFS®, HELIXATE® Fs, HELIXATE®, XYNTHA®/REFACTO AB®, HEMOFIL-M®,MONARC-M®, ALPHANATE®, KOATE-DVI®, NUWIQ® or HYATE:C®), Factor IX(Christmas Factor, e.g., a plasma derived Factor IX product such as,BERININ® P, MONOFIX® or MONONINE®; or a recombinant Factor IX productsuch as ALPHANINE SD®, ALPROLIX®, BEBULIN®, BEBULIN VH®, BENEFIX®,IXINITY®, PROFILNINE SD®, PROPLEX T®, or RIXUBIS®), Factor X(Stuart-Prower Factor), Factor XI (Plasma thromboplastin antecedent),Factor XII (Hageman (contact) factor) and Factor XIII((Fibrin-stabilizing factor/Prekallikrein (Fletcher) factor/HMWK(Fitzgerald) factor; e.g., FIBROGAMMIN® P, CORIFACT®, CLUVOT® orCLUVIAT®). In one example, the therapeutic protein is a von WillebrandFactor/FVIII complex (e.g., HUMATE-P®, HAEMATE®-P, BIOSTATE® orVONCENTO®). In an alternative example, the therapeutic protein is aprothrombin complex (e.g., BERIPLEX® P/N, CONFIDEX® or KCENTRA®). Inanother example, the therapeutic protein is a fibrinogen (e.g.,RIASTAP®, HAEMOCOMPLETTAN® P).

Von Willebrand Factor

The present disclosure provides a serum albumin variant, or functionalfragment thereof, conjugated to von Willebrand factor.

The term “von Willebrand factor” (vWF) as used herein includes naturallyoccurring (native) VWF, but also variants thereof, e.g., fragments,fusion proteins or conjugates, or sequence variants where one or moreresidues have been inserted, deleted or substituted, retaining thebiological activity of naturally occurring vWF. For the purposes ofnomenclature only and not limitation, exemplary sequences of humannative vWF are set out in NCBI Ref Seq ID: NP_000543.2 and SEQ ID NO: 6.The skilled person will appreciate that the native vWF comprisesmultiple domains. For the purposes of the present disclosure, thefollowing annotations have been prescribed:D1-D2-D′-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK, wherein the D′ domainconsists of amino acids 764 to 865 of SEQ ID NO: 6; the D3 domainconsists of amino acids 866 to 1242 of SEQ ID NO: 6; and the C1 domainconsists of amino acids 2255 to 2328 of SEQ ID NO: 6.

In one example, the vWF is a modified or mutant or variant vWF. Forexample, the modified vWF for use in the present disclosure comprises aD′D3 domain and modified forms thereof, such as truncated or mutatedforms thereof. For example, the modified vWF comprises amino acids 764to 1242 of SEQ ID NO: 6.

In one example, the von Willebrand factor is a recombinant vonWillebrand factor.

Soluble Complement Inhibitors

The present disclosure provides a serum albumin variant, or functionalfragment thereof, conjugated to a soluble complement inhibitor.

The complement system is comprised of a number of cell-surface andsoluble proteins that play a role in elimination of foreignmicroorganisms, whilst protecting the host from complement-relateddamage. The three pathways of the complement system include theclassical pathway (involving C1q, C1r, C1s, C4, C2 and C3 components),the lectin pathway and the alternative pathway. Four complementreceptors have been described, CR1 (CD35), CR2 (CD21), CR3 (CD11b/CD18)and CR4 (CD11c/CD18). CR1 is a principal regulator of the activation ofthe complement system of plasma proteins.

In one example, the serum albumin variant, or functional fragmentthereof, is conjugated to a soluble complement inhibitor, or modified(i.e., variant) form thereof.

Suitable complement inhibitors for use in the present disclosure will beapparent to the skilled person and include, for example, Factor I, (fI),Factor H (fH), C4b-binding protein (C4 bp), soluble CD55 (decayaccelerating factor (DAF)), C1-inhibitor (C1-INH or C1 esteraseinhibitor); soluble CD35 (sCR1); soluble CD46 (membrane cofactor protein(MCP)), soluble CD59 (protectin), TT30 (CR2-fH), Cobra venom factor(CVF) and a functional fragment or variant thereof.

In one example, the complement inhibitor is a soluble complementreceptor 1 (sCR1), also known as complement receptor type 1; CD35; C3BR;C3b/C4b receptor, and TP10. For example, the soluble complementinhibitor for use in the present disclosure is a modified or variantsCR1. sCR1 and variant sCR1 molecules will be apparent to the skilledperson and are described, for example, in WO1991016437, WO1994000571 andWO1997031944.

In one example, the complement inhibitor is a C1-inhibitor (C1-INH),also known as C1 esterase inhibitor, serpin family G member 1(SERPING1), HAE1, HAE2, C1NH and C1IN. For example, the complementinhibitor for use in the present disclosure is a modified or variantC1-INH. In one example, the C1-INH is plasma-derived C1-INH. In anotherexample, the C1-INH is recombinant C1-INH. C1-INH and variant C1-INHmolecules will be apparent to the skilled person and include, forexample, Berinert®. Other suitable C1-INH and variant C1-INH moleculesare described, for example, in WO2016070156.

Toxins

The present disclosure provides a serum albumin variant, or functionalfragment thereof, conjugated to a toxin.

Suitable toxins for use in the present disclosure will be apparent tothe skilled person and include, for example, ricin, abrin, diphtheriatoxin, tetanus toxoid, Pseudomonas exotoxin A (PE), and ribosomalinactivating proteins such as gelonin, pokeweed antiviral protein andsaporin.

Chemotherapy Compounds

The present disclosure provides a serum albumin variant, or functionalfragment thereof, conjugated to a chemotherapy compound.

Suitable chemotherapy compounds for use in the present disclosure willbe apparent to the skilled person and include, for example, caboplatin,cisplatin, cyclophosphamide, docetaxal, doxorubicin, erlotinib,etoposide, fluorouracil, irinotecan, methotrexate, paclitaxel,topotecan, vincristine, vinblastine, methotrexate, 1-asparaginase,vincristine, doxorubicin, danorubicin, cytarabine, idarubicin,mitoxantrone, cyclophosphamide, fludarabine, chlorambucil andderivatives thereof.

Antibodies or Antigen Binding Fragments

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to an antibody or antigenbinding fragment thereof.

Exemplary antibodies or antigen binding fragments thereof for use in thepresent disclosure are described herein or known in the art and include:

-   -   a humanized antibody or fragment thereof, e.g., a protein        comprising a human-like variable region, which includes CDRs        from an antibody from a non-human species (e.g., mouse or rat or        non-human primate) grafted onto or inserted into FRs from a        human antibody (e.g., produced by methods described in U.S. Pat.        Nos. 5,225,539, 6,054,297, 7,566,771 or U.S. Pat. No. 5,585,089)    -   a human antibody or fragment thereof, e.g., antibodies having        variable and, optionally, constant antibody regions found in        humans, e.g. in the human germline or somatic cells or from        libraries produced using such regions. The “human” antibodies        can include amino acid residues not encoded by human sequences,        e.g. mutations introduced by random or site directed mutations        in vitro (e.g., produced by methods described in U.S. Pat. No.        5,565,332) and affinity matured forms of such antibodies.    -   a synhumanized antibody or fragment thereof, e.g., an antibody        that includes a variable region comprising FRs from a New World        primate antibody variable region and CDRs from a non-New World        primate antibody variable region (e.g., produced by methods        described in WO2007019620).    -   a primatized antibody or fragment thereof, e.g., an antibody        comprising variable region(s) from an antibody generated        following immunization of a non-human primate (e.g., a        cynomolgus macaque) (e.g., produced by methods described in U.S.        Pat. No. 6,113,898).    -   a chimeric antibody or chimeric antigen binding fragment, e.g.,        an antibody or fragment in which one or more of the variable        domains is from a particular species (e.g., murine, such as        mouse or rat) or belonging to a particular antibody class or        subclass, while the remainder of the antibody or fragment is        from another species (such as, for example, human or non-human        primate) or belonging to another antibody class or subclass        (e.g., produced by methods described in U.S. Pat. Nos.        6,331,415; 5,807,715; 4,816,567 and 4,816,397).    -   a deimmunized antibody or antigen binding fragment thereof,        e.g., antibodies and fragments that have one or more epitopes,        e.g., B cell epitopes or T cell epitopes removed (i.e., mutated)        to thereby reduce the likelihood that a subject will raise an        immune response against the antibody or protein (e.g., as        described in WO2000034317 and WO2004108158).    -   a bispecific antibody or fragment thereof, e.g., an antibody        comprising two types of antibodies or antibody fragments (e.g.,        two half antibodies) having specificities for different antigens        or epitopes (e.g., as described in U.S. Pat. No. 5,731,168).

Additional exemplary antibody fragments for use in the presentdisclosure are described herein or known in the art and include:

-   -   single-domain antibodies (domain antibody or dAb), e.g., a        single polypeptide chain comprising all or a portion of the        heavy chain variable domain of an antibody.    -   a diabody, triabody, tetrabody or higher order protein complex        (e.g., as described in WO98/044001 and/or WO94/007921).    -   single chain Fv (scFv) fragments, e.g., a fragment comprising        V_(H) and V_(L) regions in a single polypeptide chain and a        polypeptide linker between the V_(H) and V_(L) which enables the        scFv to form the desired structure for antigen binding (i.e.,        for the V_(H) and V_(L) of the single polypeptide chain to        associate with one another to form a Fv).    -   a half-antibody or a half-molecule, e.g., a protein comprising a        single heavy chain and a single light chain.

The present disclosure also contemplates other antibodies and antibodyfragments, such as:

-   -   (i) minibodies, e.g., as described in U.S. Pat. No. 5,837,821;    -   (ii) heteroconjugate proteins, e.g., as described in U.S. Pat.        No. 4,676,980;    -   (iii) heteroconjugate proteins produced using a chemical        cross-linker, e.g., as described in U.S. Pat. No. 4,676,980; and    -   (iv) Fab₃ (e.g., as described in EP19930302894).

Protein Scaffolds

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a protein scaffold. Forexample, the protein scaffold is an immunoglobulin or immunoglobulinfragment.

Suitable protein scaffolds for use in the present disclosure aredescribed herein or will be apparent to the skilled person and include:

-   -   heavy chain immunoglobulins, e.g., immunoglobulins (e.g.,        antibodies) that do not comprise a light chain (e.g., as        described in WO9404678, WO9749805 and WO 9749805).    -   V-like proteins, e.g., a T-cell receptor comprising two        V-domains that combine into a structure similar to the Fv module        of an antibody (e.g., as described in Novotny et al., Proc Natl        Acad Sci USA 88: 8646-8650, 1991, WO1999045110 or WO2011107595).    -   adnectins e.g., an immunoglobulin based on the tenth fibronectin        type III (10Fn3) domain of human fibronectin in which the loop        regions are altered to confer antigen binding (e.g., as        described in US20080139791 or WO2005056764).    -   anticalins e.g., an immunoglobulin derived from lipocalins        having a rigid β-sheet secondary structure with a plurality of        loops at the open end of the conical structure which can be        engineered to bind to an antigen (e.g., as described in U.S.        Pat. No. 7,250,297 or US20070224633).    -   an affibody e.g., a scaffold derived from the Z domain (antigen        binding domain) of Protein A of Staphylococcus aureus which can        be engineered to bind to antigen (e.g., as described in        EP1641818).    -   an avimer, e.g., multidomain proteins derived from the A-domain        scaffold family (e.g., as described in WO2002088171).    -   Designed Ankyrin Repeat Protein (DARPin), e.g., derived from the        Ankyrin family of proteins that mediate attachment of integral        membrane proteins to the cytoskeleton that can be engineered to        bind different target antigens (e.g., as described in        US20040132028).        Small Molecules

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a small molecule. Such asmall molecule may be isolated from a library. Chemical small moleculelibraries are available commercially or alternatively may be generatedusing methods known in the art, such as, for example, those described inU.S. Pat. No. 5,463,564.

Techniques for synthesizing small organic compounds will varyconsiderably depending upon the compound, however such methods will beknown to those skilled in the art.

In one example, informatics is used to select suitable chemical buildingblocks from known compounds, for producing a combinatorial library. Forexample, QSAR (Quantitative Structure Activity Relationship) modelingapproach uses linear regressions or regression trees of compoundstructures to determine suitability. The software of the ChemicalComputing Group, Inc. (Montreal, Canada) uses high-throughput screeningexperimental data on active as well as inactive compounds, to create aprobabilistic QSAR model, which is subsequently used to select leadcompounds. The Binary QSAR method is based upon three characteristicproperties of compounds that form a “descriptor” of the likelihood thata particular compound will or will not perform a required function:partial charge, molar refractivity (bonding interactions), and log P(lipophilicity of molecule). Each atom has a surface area in themolecule and it has these three properties associated with it. All atomsof a compound having a partial charge in a certain range are determinedand the surface areas (Van der Walls Surface Area descriptor) aresummed. The binary QSAR models are then used to make activity models orADMET models, which are used to build a combinatorial library.Accordingly, lead compounds identified in initial screens can be used toexpand the list of compounds being screened to thereby identify highlyactive compounds.

Nucleic Acid-Based Agents

In one example, the present disclosure provides a serum albumin variant,or functional fragment thereof, conjugated to a nucleic acid-basedagent. Suitable agents will be apparent to the skilled person andinclude, for example, an antisense oligonucleotide, a short hairpin RNA(shRNA), siRNA, an interfering RNA (RNAi), a ribozyme, a microRNA and aDNAzyme.

Antisense Oligonucleotides

In one example, the nucleic acid-based agent is an antisenseoligonucleotide or antisense nucleic acid.

The terms “antisense oligonucleotide” or “antisense nucleic acid” shallbe taken to mean a DNA or RNA or derivative thereof (e.g., LNA or PNA),or combination thereof that is complementary to at least a portion of aspecific mRNA molecule encoding a polypeptide as described herein in anyexample of the disclosure and capable of interfering with apost-transcriptional event such as mRNA translation. The use ofantisense methods is known in the art (see for example, Hartmann andEndres (editors), Manual of Antisense Methodology, Kluwer (1999)).

An antisense nucleic acid of the disclosure will hybridize to a targetnucleic acid under physiological conditions. Antisense nucleic acidsinclude sequences that correspond to structural genes or coding regionsor to sequences that effect control over gene expression or splicing.For example, the antisense nucleic acid may correspond to the targetedcoding region of a nucleic acid, or the 5′-untranslated region (UTR) orthe 3′-UTR or combination of these. It may be complementary in part tointron sequences, which may be spliced out during or aftertranscription, for example only to exon sequences of the target gene.The length of the antisense sequence should be at least 19 contiguousnucleotides, for example, at least 50 nucleotides, such as at least 100,200, 500 or 1000 nucleotides of a nucleic acid. The full-length sequencecomplementary to the entire gene transcript may be used. The length canbe 100-2000 nucleotides. The degree of identity of the antisensesequence to the targeted transcript should be at least 90%, for example,95-100%.

Catalytic Nucleic Acid

In one example, the nucleic acid-based agent is a catalytic nucleicacid.

The term “catalytic nucleic acid” refers to a DNA molecule orDNA-containing molecule (also known in the art as a “deoxyribozyme” or“DNAzyme”) or a RNA or RNA-containing molecule (also known as a“ribozyme” or “RNAzyme”) which specifically recognizes a distinctsubstrate and catalyzes the chemical modification of this substrate. Thenucleic acid bases in the catalytic nucleic acid can be bases A, C, G, T(and U for RNA).

Typically, the catalytic nucleic acid contains an antisense sequence forspecific recognition of a target nucleic acid, and a nucleic acidcleaving enzymatic activity (also referred to herein as the “catalyticdomain”). The types of ribozymes that are useful in this disclosure area hammerhead ribozyme and a hairpin ribozyme.

RNA Interference

In one example, the nucleic acid-based agent is a small interfering RNA(“siRNA”) molecule.

RNA interference (RNAi) is useful for specifically inhibiting productionof a particular protein. Without being limited by theory, thistechnology relies on the presence of dsRNA molecules that contain asequence that is essentially identical to the mRNA of the gene ofinterest or part thereof. Conveniently, dsRNA can be produced from asingle promoter in a recombinant vector host cell, where the sense andanti-sense sequences are flanked by an unrelated sequence which enablesthe sense and anti-sense sequences to hybridize to form the dsRNAmolecule with the unrelated sequence forming a loop structure. Thedesign and production of suitable dsRNA molecules for the presentdisclosure is well within the capacity of a person skilled in the art.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides, such as at least 30 or 50nucleotides, for example at least 100, 200, 500 or 1000 nucleotides. Thefull-length sequence corresponding to the entire gene transcript may beused. The lengths can be 100-2000 nucleotides. The degree of identity ofthe sense and antisense sequences to the targeted transcript should beat least 85%, for example, at least 90% such as, 95-100%.

Exemplary small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. For example, the siRNA sequencecommences with the dinucleotide AA, comprises a GC-content of about30-70% (for example, 30-60%, such as 40-60% for example about 45%-55%),and does not have a high percentage identity to any nucleotide sequenceother than the target in the genome of the subject in which it is to beintroduced, for example as determined by standard BLAST search.

Assaying Activity of a Serum Albumin Variant

Serum albumin variants of the present disclosure are readily screenedfor biological activity, e.g., as described below.

Determining Affinity

Optionally, the dissociation constant (Kd) or association constant (Ka)or affinity constant (K_(D)) of a serum albumin variant, or functionalfragment thereof, is determined.

Affinity measurements can be determined by standard methodology, forexample, immunoassays, surface plasmon resonance (SPR; e.g., usingBIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, N.J.)(Rich and Myszka Curr. Opin. Biotechnol 11: 54, 2000; EnglebienneAnalyst. 123: 1599, 1998), isothermal titration calorimetry (ITC) orother kinetic interaction assays known in the art.

In some examples, the serum albumin variant, or functional fragmentthereof has a similar K_(D) or an improved K_(D) (i.e., a K_(D) valuelower than) than a serum albumin set forth in SEQ ID NO: 1.

Binding affinity for FcRn can also be determined non-quantitativelyusing flow cytometry. For example, CHO cells stably expressing the serumalbumin variant, or functional fragment thereof, are stained withalexa-488 labeled FcRn/β2m (to detect target binding) and anti-myc-alexa647 (to detect expression) at acidic (pH 5.5) and neutral (pH 7.4) pHand analysed by flow cytometry. Relative binding to FcRn/β2m isdetermined, for example, by calculating mean fluorescence intensityrelative to an unmodified serum albumin (e.g., as set forth in SEQ IDNO: 1).

Determining Half-Life

Serum albumin variants, or functional fragments thereof, encompassed bythe present disclosure have an improved half-life, e.g., are modified toextend their half-life compared to a serum albumin set forth in SEQ IDNO: 1 (i.e., a serum albumin that is unmodified). Methods fordetermining a serum albumin variant, or functional fragment thereof,with an increased half-life will be apparent to the skilled person. Forexample, the ability of a serum albumin variant, or functional fragmentthereof, to bind to a neonatal Fc receptor (FcRn) is assessed. In thisregard, increased binding affinity for FcRn increases the serumhalf-life of the serum albumin variant, or functional fragment thereof(see for example, Kim et al., Eur J Immunol., 24:2429, 1994).

The half-life of a serum albumin variant, or functional fragmentthereof, of the disclosure can also be measured by pharmacokineticstudies, e.g., according to the method described by Kim et al, Eur J ofImmunol 24:542, 1994. According to this method protein is injectedintravenously into mice and its plasma concentration is periodicallymeasured as a function of time, for example at 3 minutes to 72 hoursafter the injection. The clearance curve thus obtained should bebiphasic, that is, an alpha phase and beta phase. For the determinationof the in vivo half-life of the protein, the clearance rate inbeta-phase is calculated and compared with that of the wild type orunmodified protein (i.e., as set forth in SEQ ID NO: 1).

In vitro Cellular Assays

Various in vitro assays are available to assess the ability of a serumalbumin variant, or functional fragment thereof, to treat a disease orcondition described herein.

In one example, the uptake and recycling of the serum albumin variant,or functional fragment thereof, is tested in an in vitro cellular assay.

Methods of assessing cellular uptake and recycling are known in the artand/or exemplified herein. For example, fluorescently labelled serumalbumin variant is incubated with cells expressing the human FcRnreceptor on the cell surface. After addition of the labelled serumalbumin variant the progress of the protein recycling can be tracked andcompared to a non-modified serum albumin protein by methods includingflow cytometry and fluorescence microscopy (for example, confocalfluorescence microscopy). Changes to the normal recycling pathway for aparticular serum albumin variant can be identified and characterised.

Serum albumin variants, or functional fragments thereof, that are foundto be effectively recycled are identified as variants of the presentdisclosure.

Pharmacokinetic Analysis

In one example, the pharmacokinetic (PK) properties of the serum albuminvariant, or functional fragment thereof, will be assessed.

Methods of assessing the PK properties are known in the art and/or areexemplified herein. For example, serum albumin variants are injectedinto transgenic mice expressing human FcRn receptor or other suitablemammalian hosts (e.g. rats, cynomolgus monkeys). In one example, thetransgenic mice expressing human FcRn receptor are “hFcRn Tg32”homozygous mice (i.e., B6. Cg-FcgrttmlDcr Tg(FCGRT)32Dcr/DcrJ; TheJackson Laboratory stock number 014565; or as described in Roopenian etal., J. Immunol 2003; 170:3528-3533). Plasma levels of serum albuminwill be assessed using ELISA using commercially available methods.

Pharmaceutical Compositions

Suitably, in compositions or methods for administration of the serumalbumin variant, or functional fragment thereof, of the disclosure to asubject, the serum albumin conjugate of the present disclosure (i.e.,the serum albumin variant, or functional fragment thereof conjugated toa compound) is combined with a pharmaceutically acceptable carrier as isunderstood in the art. Accordingly, one example of the presentdisclosure provides a composition (e.g., a pharmaceutical composition)comprising the serum albumin conjugate of the disclosure combined with apharmaceutically acceptable carrier.

In general terms, by “carrier” is meant a solid or liquid filler,binder, diluent, encapsulating substance, emulsifier, wetting agent,solvent, suspending agent, coating or lubricant that may be safelyadministered to any subject, e.g., a human. Depending upon theparticular route of administration, a variety of acceptable carriers,known in the art may be used, as for example described in Remington'sPharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991).

A serum albumin conjugate of the present disclosure is useful forparenteral, topical, oral, or local administration, aerosoladministration, or transdermal administration, for prophylactic or fortherapeutic treatment. In one example, the serum albumin conjugate isadministered parenterally, such as subcutaneously or intravenously. Forexample, the serum albumin conjugate is administered intravenously.

Formulation of a serum albumin conjugate to be administered will varyaccording to the route of administration and formulation (e.g.,solution, emulsion, capsule) selected. An appropriate pharmaceuticalcomposition comprising a serum albumin conjugate to be administered canbe prepared in a physiologically acceptable carrier. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. A variety of appropriate aqueous carriers areknown to the skilled artisan, including water, buffered water, bufferedsaline, polyols (e.g., glycerol, propylene glycol, liquid polyethyleneglycol), dextrose solution and glycine. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers (See, generally, Remington's Pharmaceutical Science, 16thEdition, Mack, Ed. 1980). The compositions can optionally containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents and toxicity adjusting agents, for example, sodium acetate,sodium chloride, potassium chloride, calcium chloride and sodiumlactate. The serum albumin conjugate can be stored in the liquid stageor can be lyophilized for storage and reconstituted in a suitablecarrier prior to use according to art-known lyophilization andreconstitution techniques.

Conditions to be Treated

As discussed herein, the present disclosure provides a method oftreating or preventing a disease or condition in a subject, the methodcomprising administering the serum albumin conjugate of the presentdisclosure or the composition of the present disclosure to a subject inneed thereof. In one example, the present disclosure provides a methodof treating a disease or condition in a subject in need thereof.

The present disclosure also provides for use of a serum albuminconjugate of the present disclosure for treating or preventing a diseaseor condition in a subject comprising administering the serum albuminconjugate of the present disclosure or the composition of the presentdisclosure to a subject in need thereof. In one example, the presentdisclosure provides for use of a serum albumin conjugate of the presentdisclosure for treating a disease or condition in a subject in needthereof.

In one example, the disease or condition is a bleeding disorder.

In one example, the subject suffers from a bleeding disorder. Thebleeding disorder can be inherited or acquired. For example, a subjectsuffering from a bleeding disorder has suffered a symptom of a bleedingdisorder, such as:

Easy bruising;

Bleeding gums;

Heavy bleeding from small cuts or dental work;

Unexplained nosebleeds;

Heavy menstrual bleeding;

Bleeding into joints; and/or

Excessive bleeding following surgery.

In one example, the subject is at risk of developing a bleedingdisorder. A subject is at risk if he or she has a higher risk ofdeveloping a bleeding disorder than a control population. The controlpopulation may include one or more subjects selected at random from thegeneral population (e.g., matched by age, gender, race and/or ethnicity)who have not suffered from or have a family history of angina, strokeand/or heart attack. A subject can be considered at risk for a bleedingdisorder if a “risk factor” associated with a bleeding disorder is foundto be associated with that subject. A risk factor can include anyactivity, trait, event or property associated with a given disorder, forexample, through statistical or epidemiological studies on a populationof subjects. A subject can thus be classified as being at risk for ableeding disorder even if studies identifying the underlying riskfactors did not include the subject specifically. For example, a subjectwho has excessive bleeding is at risk of developing a bleeding disorderbecause the frequency of a bleeding disorder is increased in apopulation of subjects who have excessive bleeding as compared to apopulation of subjects who do not.

In one example, the subject is at risk of developing a bleeding disorderand the serum albumin conjugate is administered before or after theonset of symptoms of a bleeding disorder. In one example, the serumalbumin conjugate is administered before the onset of symptoms of ableeding disorder. In one example, the serum albumin conjugate isadministered after the onset of symptoms of a bleeding disorder. In oneexample, the serum albumin conjugate of the present disclosure isadministered at a dose that alleviates or reduces one or more of thesymptoms of a bleeding disorder in a subject at risk.

The methods of the present disclosure can be readily applied to any formof bleeding disorder in a subject.

A method of the present disclosure may also include co-administration ofthe serum albumin conjugate according to the disclosure together withthe administration of another therapeutically effective agent for theprevention or treatment of a bleeding disorder.

In one example, the serum albumin conjugate of the disclosure is used incombination with at least one additional known compound or therapeuticprotein which is currently being used or is in development forpreventing or treating bleeding disorders. Compounds currently used inthe treatment of bleeding disorders are known in the art. Exemplarytherapeutic proteins may be plasma derived from a donor or a recombinantprotein. For example, the therapeutic protein is a plasma derived orrecombinant coagulation factor protein. For example, the therapeuticprotein is selected from the group consisting of Factor I, Factor II((prothrombin)/thrombin), Factor III, Factor V, Factor VII, Factor VIIa(e.g., NOVOSEVEN®), Factor VIII (such as a single chain recombinantFactor VIII, e.g., as described in Zollner et al., Thromb Res.132:280-287, 2013; or a plasma derived Factor VIII product, such asFEIBA®, MONOCLATE-P®, or BIOSTATE®; or a recombinant Factor VIIIproduct, such as ADVATE®, ELOCTATE®, RECOMBINATE®, KOGENATE FS®,HELIXATE® Fs, HELIXATE®, XYNTHA®/REFACTO AB®, HEMOFIL-M®, MONARC M®,ALPHANATE®, KOATE-DVI®, NUWIQ® or HYATE:C®), Factor IX (e.g., a plasmaderived Factor IX product such as, BERININ® P, MONOFIX® or MONONINE®; ora recombinant Factor IX product such as ALPHANINE SD®, ALPROLIX®,BEBULIN®, BEBULIN VH®, BENEFIX®, IXINITY®, PROFILNINE SD®, PROPLEX T®,or RIXUBIS®), Factor X, Factor XI, Factor XII and Factor XIII (e.g.,FIBROGAMMIN® P, CORIFACT®, CLUVOT® or CLUVIAT®). In one example, thetherapeutic protein is a von Willebrand Factor/FVIII complex (e.g.,HUMATE-P®, HAEMATE®-P, BIOSTATE® or VONCENTO®). In an alternativeexample, the therapeutic protein is a prothrombin complex (e.g.,BERIPLEX® P/N, CONFIDEX® or KCENTRA®). In another example, thetherapeutic protein is a fibrinogen (e.g., RIASTAP®, HAEMOCOMPLETTAN®P). In one example, the therapeutic protein is a modified form of acoagulation factor, e.g., as described herein.

As will be apparent from the foregoing, the present disclosure providesmethods of concomitant therapeutic treatment of a subject, comprisingadministering to a subject in need thereof an effective amount of afirst agent and a second agent, wherein the first agent is a serumalbumin conjugate of the present disclosure, and the second agent isalso for the prevention or treatment of a bleeding disorder.

As used herein, the term “concomitant” as in the phrase “concomitanttherapeutic treatment” includes administering a first agent in thepresence of a second agent. A concomitant therapeutic treatment methodincludes methods in which the first, second, third or additional agentsare co-administered. A concomitant therapeutic treatment method alsoincludes methods in which the first or additional agents areadministered in the presence of a second or additional agent, whereinthe second or additional agent, for example, may have been previouslyadministered. A concomitant therapeutic treatment may be executedstep-wise by different actors. For example, one actor may administer toa subject a first agent and as a second actor may administer to thesubject a second agent and the administering steps may be executed atthe same time, or nearly the same time, or at distant times, so long asthe first agent (and/or additional agents) are after administration inthe presence of the second agent (and/or additional agents). The actorand the subject may be the same entity (e.g. a human)

The optimum concentration of the active ingredient(s) in the chosenmedium can be determined empirically, according to procedures known tothe skilled artisan, and will depend on the ultimate pharmaceuticalformulation desired.

The dosage ranges for the administration of the serum albumin conjugateof the disclosure are those large enough to produce the desired effect.For example, the composition comprises an effective amount of the serumalbumin conjugate. In one example, the composition comprises atherapeutically effective amount of the serum albumin conjugate. Inanother example, the composition comprises a prophylactically effectiveamount of the serum albumin conjugate.

The dosage should not be so large as to cause adverse side effects, suchas paradoxical bleedings and development of inhibitors. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient and can be determined by one of skill in the art. Thedosage can be adjusted by the individual physician in the event of anycomplication.

Dosage can vary from about 0.1 mg/kg to about 300 mg/kg, e.g., fromabout 0.2 mg/kg to about 200 mg/kg, such as, from about 0.5 mg/kg toabout 20 mg/kg, in one or more dose administrations daily, for one orseveral days.

In some examples, the serum albumin conjugate is administered at aninitial (or loading) dose which is higher than subsequent (maintenancedoses). For example, the v is administered at an initial dose of betweenabout 10 mg/kg to about 30 mg/kg. The binding protein is thenadministered at a maintenance dose of between about 0.0001 mg/kg toabout 10 mg/kg. The maintenance doses may be administered every 7-35days, such as, every 7 or 14 or 28 days.

In some examples, a dose escalation regime is used, in which a serumalbumin conjugate is initially administered at a lower dose than used insubsequent doses. This dosage regime is useful in the case of subject'sinitially suffering adverse events

In the case of a subject that is not adequately responding to treatment,multiple doses in a week may be administered. Alternatively, or inaddition, increasing doses may be administered.

A subject may be retreated with the serum albumin conjugate, by beinggiven more than one exposure or set of doses, such as at least about twoexposures of the binding protein, for example, from about 2 to 60exposures, and more particularly about 2 to 40 exposures, mostparticularly, about 2 to 20 exposures.

In one example, any retreatment may be given when signs or symptoms ofdisease return, e.g., a bleeding episode.

In another example, any retreatment may be given at defined intervals.For example, subsequent exposures may be administered at variousintervals, such as, for example, about 24-28 weeks or 48-56 weeks orlonger. For example, such exposures are administered at intervals eachof about 24-26 weeks or about 38-42 weeks, or about 50-54 weeks.

In the case of a subject that is not adequately responding to treatment,multiple doses in a week may be administered. Alternatively, or inaddition, increasing doses may be administered.

In another example, for subjects experiencing an adverse reaction, theinitial (or loading) dose may be split over numerous days in one week orover numerous consecutive days.

Administration of a serum albumin conjugate according to the methods ofthe present disclosure can be continuous or intermittent, depending, forexample, on the recipient's physiological condition, whether the purposeof the administration is therapeutic or prophylactic, and other factorsknown to skilled practitioners. The administration may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses, e.g., either during or after development of a condition.

Kits and Other Compositions of Matter

Another example of the disclosure provides kits containing a serumalbumin conjugate of the present disclosure useful for the treatment orprevention of a bleeding disorder as described above.

In one example, the kit comprises (a) a container comprising a serumalbumin conjugate optionally in a pharmaceutically acceptable carrier ordiluent; and (b) a package insert with instructions for treating orpreventing a bleeding disorder in a subject.

In one example, the kit comprises (a) at least one serum albuminconjugate; (b) instructions for using the kit in treating or preventingthe bleeding disorder in the subject; and (c) optionally, at least onefurther therapeutically active compound or drug.

In accordance with this example of the disclosure, the package insert ison or associated with the container. Suitable containers include, forexample, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds or contains a composition that is effective for treating orpreventing a bleeding disorder and may have a sterile access port (forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is the serum albumin conjugate. Thelabel or package insert indicates that the composition is used fortreating a subject eligible for treatment, e.g., one having orpredisposed to developing a bleeding disorder, with specific guidanceregarding dosing amounts and intervals of the serum albumin conjugateand any other medicament being provided.

The kit may further comprise an additional container comprising apharmaceutically acceptable diluent buffer, such as bacteriostatic waterfor injection (BWFI), phosphate-buffered saline, Ringer's solution,and/or dextrose solution. The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, and syringes.

The kit optionally further comprises a container comprises a secondmedicament, wherein the serum albumin conjugate is a first medicament,and which article further comprises instructions on the package insertfor treating the subject with the second medicament, in an effectiveamount. The second medicament may be a therapeutic protein set forthabove.

The present disclosure includes the following non-limiting Examples.

EXAMPLES Example 1: Generation of Human Serum Albumin Variants

A library of randomly-mutated variants of human serum albumin (HSA) wasgenerated using standard error-prone PCR of human serum albuminTruncation and frameshift variants were removed using intein-based openreading frame (ORF) selection, as previously described in Gerth et al.(Protein Eng Des Sel 2004 17 (7):595-602).

HSA variant clones were then subcloned and transfected into Flp-In™ CHOcells (Invitrogen) to generate a library of transfected stable celllines, each containing a single copy of a mutant albumin fusion protein.Briefly, a Flp-In™ CHO host cell line containing an integrated Flprecombination target (PRT) site was obtained. The library of HSAvariants was cloned into the pcDNA5/FRT expression plasmid in frame withthe platelet derived growth factor receptor (PDGFR) transmembrane domainand a Myc tag, under the control of the human CMV promoter. ThepcDNA5/FRT vector containing the HSA library and a pOG44 plasmid, whichconstitutively expresses the Flp recombinase under the control of thehuman CMV promoter, were co-transfected into the Flp-In™ host cell line,resulting in all transfected cells that successfully underwentFlp-catalysed recombination harboring one copy of the mutant albuminfusion protein. Expression of the HSA variants as fusion proteins withthe PDGFR transmembrane region allowed extracellular display ofmembrane-tethered variants on the surface of mammalian cells. Stablytransfected cells were grown for approximately 14 days in selectionmedia to remove non-transfected cells.

Example 2: Identification of Residues Associated with Increased Affinityfor FcRn/β2m

To determine the binding affinity of the HSA variants to neonatal Fcreceptor (FcRn/β2m), individual pools of CHO cells stably expressing HSAvariants were stained with alexa-488 labeled FcRn/β2m (10 μg/ml at pH5.4and 100 μg/ml at pH7.3; to detect target binding) and anti-myc-alexa 647(to detect expression of the construct) at pH 5.4 and pH 7.3 andanalysed by flow cytometry. The amount of labelled FcRn bound to cellsexpressing each HSA variant was quantified as the geometric meanfluorescence intensity (geoMFI) data of the stained cells, backgroundfluorescence due to non-specific binding of the labelled FcRn to thecells was subtracted and relative binding to FcRn/β2m was determinedcompared to wild-type HSA and HSA variant K573Y (described in Andersonet al. (2014) J Biol Chem, 289:13492).

Two clones (clone 3.9 and clone 5.6) were selected for further analysisbased on affinity and individual mutations within each clone weredissected out to determine the contribution of each mutation to theoverall affinity of the parent clone.

FcRn binding to each of the HSA variants was measured by flow cytometryin two independent experiments. The amount of labelled FcRn bound tocells expressing a HSA variant was quantified as the geometric meanfluorescence intensity (geoMFI) of the stained cells. Backgroundfluorescence due to non-specific binding of the labelled FcRn to thecells was subtracted from the value obtained. Improved binding toFcRn/β2m over wild-type was observed at pH5.4 for positive control HSAvariant K573Y, as well as HSA variants with the single substitutionsQ522L, G572R and A552V. As shown in Table 1, a number of clones showedno improvement in binding over wild-type, including T133I, K162N, K317E,F377I, I388T, F157L, P282S and Y452S. None of the clones showed anyappreciable binding to FcRn/β2m at pH 7.3.

Example 3: Generation of HSA Variants with Single Amino AcidSubstitutions

Amino acid residues Q522, G572 and A552 were selected as residues ofinterest. A panel of HSA variants was generated with single substitutionof every amino acid, except cysteine, at residue Q522 or G572 and stablyexpressed in CHO cells. Binding of HSA variants to FcRn/β2m wasdetermined as described above by flow cytometry in four independentexperiments. The amount of labelled FcRn bound to cells expressing eachHSA variant was quantified as the geometric mean fluorescence intensity(geoMFI) data of the stained cells. Background fluorescence due tonon-specific binding of the labelled FcRn to the cells was subtractedand upper and lower confidence intervals (95% CI) for eachexperimentally determined mean value calculated.

The FcRn binding intensity (geoMFI) of all HSA variants at both pH 5.5and pH 7.4 was normalized separately to adjust for systematicdifferences in measured intensities between experiments. For both pHconditions the replicate measurements of the binding of a given HSAvariant were adjusted to a mean value across replicates (similar tointensity normalization of microarray data as described in Dudoit et al.2002 Statistica Sinica 12, 111-139). For measurement of FcRn binding atpH 5.5, the replicates had clear non-linear differences across the rangeof geoMFI values measured for all the tested variants. Quadraticfunctions were fit by robust regression to each replicate's bindingstrength versus the mean (across the 4 replicates) binding strength,with all geoMFI values on a log 2 scale. Through the adjustment, eachpredicted experiment-specific binding strength was replaced by theacross-experiments mean binding strength, while each variant's residualremained the same. For measurement of FcRn binding at pH 7.4, linearfunctions were fit (i.e., the adjustment step is equivalent tosubtracting an experiment specific (small) intercept constant plusdividing by an experiment specific scaling constant).

Nine Q522 variants demonstrated statistically significant increasedFcRn/β2m binding at acidic pH 5.5: Q522G, Q522I, Q522K, Q522M, Q522F,Q522W, Q522Y, Q522V and Q522L relative to wild-type HSA (Table 2). SixG572 variants demonstrated statistically significant increased FcRn/β2mbinding at acidic pH 5.5: G572A, G572E, G572H, G572S, G572K and G572Rrelative to wild-type HSA (Table 3). No significant binding of any ofthe HSA variants to FcRn/β2m was observed at pH 7.4. Substitutions atQ522L, A552V, G572R and K573Y showed the most significant increase inbinding to FcRn/β2m at acidic pH 5.5 (Table 4) relative to wild-typeHSA.

Example 4: Generation of HSA Variants with Multiple Amino AcidSubstitutions

To test the additive or synergistic effects of the mutations on FcRn/β2mbinding, HSA variants harboring double, triple and quadruple amino acidsubstitutions were generated as previously described.

As shown in Table 5, double HSA variants Q522L/A552V, Q522L/G572R andA552V/G572R demonstrated significantly improved FcRn/β2m binding atpH5.5 over wild-type HSA and corresponding HSA variants with singleamino acid substitutions.

Double HSA variants harboring the K573Y mutation (Q522L/K573Y,A552V/K573Y and G572R/K573Y) showed significantly improved FcRn/β2mbinding at pH5.5 over wild-type HSA and the single HSA variants Q522LA552V, G572R. Double HSA variants Q552L/K573Y and A552V/K573Y but notG572R/K573Y also showed significantly improved FcRn/β2m binding at pH5.5over HSA variant K573Y.

Triple HSA variant Q522L/A522V/G572R demonstrated significantly improvedFcRn/β2m binding at pH5.5 over wild-type HSA and corresponding HSAvariants with single and double amino acid substitutions, as well asapproximately twice the level of binding compared to HSA variant K573Y(Table 5).

Triple HSA variants Q522L/A552V/K573Y and Q522L/G572R/K573Y demonstratedsignificantly improved FcRn/β2m binding at pH5.5 over wild-type HSA andcorresponding HSA variants with single amino acid substitutions, as wellas significantly improved binding over HSA variant K573Y.

Quadruple HSA variant Q522L/A552V/G572R/K573Y demonstrated significantlyimproved FcRn/β2m binding at pH5.5 over wild-type HSA and correspondingHSA variants with single amino acid substitutions. However, thequadruple variant also demonstrated significantly increased FcRn/β2mbinding at pH7.4 over wild-type HSA.

Example 5: Binding Kinetics of HSA Variants with Single, Double andTriple Amino Acid Substitutions

To test the effects of the mutations on FcRn/β2m binding, single, doubleand triple HSA variants were generated as previously described. Bindingkinetics were measured using SPR with BIAcore™ 4000 (GE Healthcare LifeSciences). Recombinant hFcRn/β2M was chemically biotinylated andtethered to a GE streptavidin sensor chip (GE Healthcare Life Sciences).Briefly, human FcRn/β2M was chemically modified at a low protein:biotinratio in PBS (i.e., minimal biotinylation) using an extended biotinlinker (Sulfo-NHS-LC-LC-Biotin) prepared at 1:0.25 molar ratio on icefor 2 hours. The reaction mix was desalted using a spin column (10000NMW) according to the manufacturer's instructions.

HSA variants were injected at concentrations of 5, 1.7, 0.6 and 0 μM.Assays were performed in 10 mM HEPES; 150 mM NaCl pH 6.0 at 37° C. inquadruplicate. The association and dissociation phases were monitoredfor 100 seconds and each cycle ended with a regeneration step atneutral, pH 7.4 for 90 seconds. Sensograms were double-subtracted fromthe reference (spot 3) and blank buffer injections within each run.

Rate constants and overall affinity rates were obtained for all HSAvariants screened against minimally biotinylated human FcRn at pH 6.Data obtained from 1.7 μM and 0.6 μM injections fit well to a 1:1binding model while data acquired from 5 μM injections were used formodel fitting due to saturated responses.

As shown in Table 6, eight Q522 variants demonstrated increased FcRn/β2mbinding at acidic pH 6.0: Q522F, Q522G, Q522M, Q522K, Q522Y, Q522I,Q522A and Q522L relative to wild-type HSA. Six G572 variantsdemonstrated increased FcRn/β2m binding at acidic pH 6.0: G572R, G572K,G572V, G572A, G572S and G572H relative to wild-type HSA (Table 6).Double HSA variants A522V/G572R, Q522L/A552V and triple HSA variantQ522L/A552V/G572R all demonstrated increased FcRn/β2m binding at acidicpH 6.0 relative to wild-type HSA (Table 6).

Example 6: Binding Kinetics of HSA Variants with Single, Double andTriple Amino Acid Substitutions

Binding kinetics of single, double and triple HSA variants on FcRn/β2mbinding was measured using SPR with BIACORE™ 8000 (GE Healthcare LifeSciences). HSA [H464Q] used as negative control.

Recombinant hFcRn/β2M was chemically biotinylated and tethered to GE'sstreptavidin sensor chip as described above. HSA variants were injectedat concentrations ranging from 2 to 0.015 μM. Assay was performed in 10mM HEPES; 150 mM NaCl (pH 6.0) at 37° C. in triplicate. The associationphase was monitored for 120 seconds and the dissociation for 240seconds. Each cycle ended with a regeneration step under basicconditions (i.e. pH 8.0) for 90 seconds. Sensograms weredouble-subtracted from the reference and blank buffer injections withineach run.

Data obtained under acidic conditions fit well to a 1:1 binding model.Rate constants and binding affinity were established as previouslydescribed.

As shown in Table 7, the majority of HSA mutants showed improvement (upto 3-fold) in binding affinity (KD) compared to the wild type HSA.Double HSA variants [Q522L/A552V], [Q522L/G572R] and [A552V/G572R]showed up to 10-fold improved binding affinity to hFcRn/β2m compared torecombinant wild-type HSA. Triple HSA variant [Q522L/A552V/G572R] hadthe strongest binding, at approximately 25 nM. Negative control HSA[H464Q] did not bind hFcRn/β2m.

Example 7: Binding Affinity of Select HSA Variants to MammalianFc12n/β2m at Acidic and Neutral pH

Single HSA variants G572R and K573Y, double HSA variants Q522L/A552V,Q522L/G572R and A552V/G572R and triple HSA variant Q522L/A552V/G572Rwere generated as previously described and binding kinetics measuredusing SPR with BIACORE™ 4000 (GE Healthcare Life Sciences).

HSA variants were diluted to 1 μg/mL and directly immobilized on twosurface sites of a carboxymethyl dextran (CM-5) sensor chip (GEHealthcare Life Sciences) using amine-coupling chemistry (NHS/EDC). HSAvariants were immobilised to values between 100 and 500RU in the outerspot (1 and 5) and 200 to 1000 RU in the inner spot (2 and 4) of eachflow cell. Spot 3 of each flow cell was activated/deactivated and usedfor reference subtraction. The flow rate of FcRn/β2m was kept constantat 30 μL/min

The binding of soluble recombinant human, cynomolgus monkey (‘cyno’),rat and mouse FcRn/β2m to immobilized HSA variants was tested at acidic(pH 6.0) and neutral pH (pH7.3). A concentration range of 2 to 0.007 μM(pH6.0) and 20 to 0.078 μM (pH7.3) was used and association anddissociation phases were monitored for 180 and 600 seconds respectively.

Regeneration of the surface was performed as previously described.Sensogram data obtained at acidic conditions fitted well to a 1:1binding model. However, sensograms obtained at pH 7.3 showed off-ratesnear the limit of detection of the instrument, requiring a steady-statebinding model for analysis.

As shown in Table 8, at pH 6.0 human and cyno FcRn/β2m showed nanomolarbinding affinity. Human and cyno FcRn/β2m showed near 20-fold improvedaffinity for HSA K573Y and 5-fold improved affinity for HSA G572Rcompared to wild-type HSA at pH 6. The affinity of double HSA variantsQ522L/A552V, Q522L/G572R and A552V/G572R was about 50 nM, about 27 nMand about 27 nM to human FcRn/β2m, respectively, and about 84 nM, 49 nMand 48 nM to cyno FcRn/β2m, respectively.

In comparison, rat and murine FcRn/β2m showed weak to no binding underthe same conditions. Rat FcRn/β2m showed 100-fold weaker affinity to HSAvariants and no binding to wild-type HSA, HSA variant Q522L/A552V andHSA variant G572R. Rat FcRn/β2m bound HSA K573Y at approximately 1 μMunder these conditions but no binding was detected for HSA G572R. MouseFcRn/β2m did not bind any of the HSA variants tested.

As shown in Table 8, at pH 7.3 human and cyno FcRn/β2m did not bindwild-type HSA and showed weak binding (>50 μM) to HSA K573Y at pH7.3.Binding affinity to other HSA variants ranged from 7 to 63 μM. Rat andmurine FcRn/β2m did not bind any of the HSA variants tested at pH 7.3.

Example 8: Pharmacokinetic (PK) Properties of HSA Variants in MiceExpressing Human FcRn

To assess the PK properties of the HSA variants, transgenic miceexpressing human FcRn receptor (“hFcRn Tg32” homozygous mice (i.e.,B6.Cg-FcgrttmlDcr Tg(FCGRT)32Dcr/DcrJ) were used. Animals of mixed sexand age (10-14 weeks old; 3 animals per time point) were intravenouslyinjected with 10 mg/kg of wild-type HSA, single HSA variant K573Y,double HSA variant Q522L/A552V or triple HSA variant Q522L/A552V/G572R.

Blood was collected and plasma levels of human serum albumin assessedusing a human albumin ELISA kit (Bethyl Laboratories, cat no. E88-129)according to the manufacturer's instructions with the exception of usingeach test article to generate an individual reference curve. Data wasaveraged for each time point prior to analysis and data was analysed bynon-linear (Marquandt-Levenberg) minimisation fitting using a customMATLAB program NCAPKfit. Model fitting was done with 1/Y{circumflex over( )}2 weighting fitting using least squares and the equation used was:PK2(t)=A*exp(−log(2)*t/T1)+B*exp(−log(2)*t/T2).

Mean residence time (MRT), area under the curve (AUC) and clearancerates were calculated using standard statistical formulae.

As shown in FIG. 1, Table 9 and Table 10 below, the pharmacokineticproperties of wildtype HSA were significantly different from all threeHSA variants, with wildtype HSA having shorter mean residence time(MRT), smaller area under the curve (AUC) and faster clearance. SingleHSA variant K573Y, double HSA variant Q522L/A552V and triple HSA variantQ522L/A552V/G572R all had significantly better pharmacokinetics by allthree criteria (i.e., MRT, AUC and clearance) compared to the wildtypeHSA.

TABLE 1 Binding of FcRn-alexa488 to HSA variants at pH 5.4 (10 μg/mLFcRn-alexa488) and pH 7.3 (100 μg/mL FcRn-alexa488) pH 5.4 pH 7.3 HSAvariant geoMFI Fold change to WT geoMFI Fold change to WT Clone 5.6 HSA[T133I/K162N/N295I/Q522L] 15931.6 9.5 24.6 1.5 (clone 5.6) HSA[T133I/K162N/Q522L] 15090.6 9.0 23 1.4 HSA [T133I/Q522L] 10839.6 6.520.1 1.2 HSA [T133I] 1265.6 0.8 18.9 1.2 HSA [K162N] 1437.6 0.9 17.4 1.1HSA [Q522L] 16468.6 9.8 17.4 1.1 HSA [WT] 1674.6 1.0 16.3 1.0 HSA[K573Y] 45011.6 26.9 15.6 1.0 Clone 3.9 HSA 34796.6 20.8 28.8 1.8[K317E/M329V/K372E/F377I/I388T/ A552V/G572R] (clone 3.9) HSA[K317E/K372E/A552V/G572R] 29479.6 17.6 29.6 1.8 HSA[T133I/Q522L/A552V/G572R] 37814.6 22.6 53.8 3.3 HSA [K317E] 1374.6 0.823.9 1.5 HSA [M329V] 1329.6 0.8 18.7 1.1 HSA [K372E] 1469.6 0.9 21.7 1.3HSA [F377I] 1483.6 0.9 9.5 0.6 HSA [I388T] 3444.6 2.1 13.9 0.9 HSA[A552V] 8082.6 4.8 17.5 1.1 HSA [G572R] 11924.6 7.1 21.6 1.3 HSA[A552V/G572R] 30527.6 18.2 32 2.0 HSA [WT] 1674.6 1.0 16.3 1.0 HSA[K573Y] 45011.6 26.9 15.6 1.0

TABLE 2 Mean and range (95% confidence interval) of binding ofFcRn-alexa488 to HSA variants at pH 5.5 and pH 7.4. pH 5.5 pH 7.4 MeangeoMFI Fold change to WT Mean geoMFI Fold change to WT HSA variant (95%CI) P value (95% CI) (95% CI) P value (95% CI) HSA[Q522A] 170.9(133.5-218.8) ns 1.07 (0.8-1.4) 17.8 (5.8-54.7) ns 0.45 (0.1-1.4)HSA[Q522R] 386.3 (253.2-589.4) ns 2.42 (1.6-3.7) 17.8 (4.0-78.7) ns 0.45(0.1-2.0) HSA[Q522N] 153.8 (131.3-180.3) ns 0.97 (0.8-1.1) 31.4(18.4-53.6) ns 0.79 (0.5-1.3) HSA[Q522D] 316.6 (262.3-382.1) ns 1.99(1.6-2.4) 22.4 (14.7-34.2) ns 0.56 (0.4-0.9) HSA[Q522E] 127.9(92.6-176.6) ns 0.8 (0.6-1.1) 29.2 (15.5-55.0) ns 0.73 (0.4-1.4)HSA[Q522G] 383.9 (317.9-463.6) (1) 2.41 (2.0-2.9) 26.4 (7.5-92.8) ns0.66 (0.2-2.3) HSA[Q522H] 147.7 (132.5-164.8) ns 0.93 (0.8-1.0) 27.2(11.7-62.9) ns 0.68 (0.3-1.6) HSA[Q522I] 666.3 (544.3-815.6) (1) 4.18(3.4-5.1) 26.3 (4.8-144.7) ns 0.66 (0.1-3.6) HSA[Q522K] 452(356.2-573.6) (1) 2.84 (2.2-3.6) 30.4 (9.9-93.8) ns 0.77 (0.2-2.4)HSA[Q522M] 586.3 (511.7-671.8) (1) 3.68 (3.2-4.2) 38 (20.8-69.5) ns 0.96(0.5-1.7) HSA[Q522F] 1379.6 (1069.9-1779.1) (2) 8.66 (6.7-11.2) 27.9(8.4-93.0) ns 0.7 (0.2-2.3) HSA[Q522P] 169.4 (144.9-198.1) ns 1.06(0.9-1.2) 29.4 (10.2-85.4) ns 0.74 (0.3-2.1) HSA[Q522S] 257.6(218.9-303.1) ns 1.62 (1.4-1.9) 32.7 (10.9-98.0) ns 0.82 (0.3-2.5)HSA[Q522T] 290.3 (239.0-352.6) ns 1.82 (1.5-2.2) 28.3 (10.3-77.7) ns0.71 (0.3-2.0) HSA[Q522W] 596.2 (406.4-874.6) (1) 3.74 (2.5-5.5) 20.2(6.5-62.3) ns 0.51 (0.2-1.6) HSA[Q522Y] 1213.6 (1074.5-1370.7) (2) 7.61(6.7-8.6) 18.6 (4.9-71.3) ns 0.47 (0.1-1.8) HSA[Q522V] 617 (477.6-797.0)(1) 3.87 (3.0-5.0) 32.8 (14.0-76.9) ns 0.82 (0.4-1.9) HSA[Q522L] 2203.6(1689.9-2873.3) (2) 13.83 (10.6-18.0) 30.3 (10.8-85.1) ns 0.76 (0.3-2.1)HSA WT 159.4 (85.6-296.7) n/a 1.0 39.8 (16.9-93.3) n/a 1.0 HSA [K573Y]29147.9 (20225.5-42006.2) (2) 182.89 (126.9-263.6) 27.6 (12.8-59.7) ns0.7 (0.3-1.5) (1) Significant difference (p < 0.01) from HSA WT; (2)Significant difference (p < 0.001) from HSA WT; ns = not significantlygreater than wild-type HSA. n/a = not applicable

TABLE 3 Mean and range (95% confidence interval) of binding ofFcRn-alexa488 to HSA variants at pH 5.5 and pH 7.4. pH 5.5 pH 7.4 MeangeoMFI Fold change to WT Mean geoMFI Fold change to WT HSA variant (95%CI) P value (95% CI) (95% CI) P value (95% CI) HSA[G572A] 536.4(419.3-686.3) (1) 3.37 (2.6-4.3) 29.8 (16.0-55.7) ns 0.75 (0.4-1.4)HSA[G572N] 85.8 (42.0-175.6) ns 0.54 (0.3-1.1) 36.4 (19.9-66.7) ns 0.92(0.5-1.7) HSA[G572D] 304.9 (235.1-395.5) ns 1.91 (1.5-2.5) 32.7(16.8-63.9) ns 0.82 (0.4-1.6) HSA[G572Q] 200.8 (108.3-372.3) ns 1.26(0.7-2.3) 40.2 (23.5-68.7) ns 1.01 (0.6-1.7) HSA[G572E] 378.7(298.2-480.8) (1) 2.38 (1.9-3.0) 28.8 (12.5-66.6) ns 0.72 (0.3-1.7)HSA[G572H] 495.1 (356.3-687.9) (1) 3.11 (2.2-4.3) 33.5 (13.5-83.4) ns0.84 (0.3-2.1) HSA[G572I] 35.2 (13.7-90.5) ns 0.22 (0.1-0.6) 28.9(11.2-74.7) ns 0.73 (0.3-1.9) HSA[G572L] 31.2 (7.4-131.1) ns 0.2(0.05-0.8) 20.3 (11.4-36.0) ns 0.51 (0.3-0.9) HSA[G572K] 9200.8(5674.0-14919.5) (2) 57.73 (35.6-93.6) 22.7 (12.9-39.7) ns 0.57(0.3-1.0) HSA[G572M] 38.2 (6.7-218.8) ns 0.24 (0.04-1.4) 24 (13.1-44.1)ns 0.6 (0.3-1.1) HSA[G572F] 25.7 (6.4-103.5) ns 0.16 (0.04-0.6) 20.6(10.3-41.2) ns 0.52 (0.3-1.0) HSA[G572P] 215.9 (20.5-2276.5) ns 1.35(0.1-14.3) 30.6 (18.9-49.7) ns 0.77 (0.5-1.2) HSA[G572S] 445.1(390.5-507.3) (1) 2.79 (2.5-3.2) 18.4 (8.4-40.6) ns 0.46 (0.2-1.0)HSA[G572T] 38 (18.1-79.8) ns 0.24 (0.1-0.5) 18.1 (4.4-74.4) ns 0.46(0.1-1.9) HSA[G572W] 34.4 (16.4-71.9) ns 0.22 (0.1-0.5) 21.2 (8.4-53.6)ns 0.53 (0.2-1.3) HSA[G572Y] 39 (15.9-95.5) ns 0.24 (0.1-0.6) 29.9(14.4-62.1) ns 0.75 (0.4-1.6) HSA[G572V] 58.2 (47.3-71.6) ns 0.37(0.3-0.4) 20.7 (8.3-51.3) ns 0.52 (0.2-1.3) HSA[G572R] 6814.2(4453.0-10427.3) (2) 42.76 (27.9-65.4) 31.7 (12.0-84.3) ns 0.8 (0.3-2.1)HSA WT 159.4 (85.6-296.7) n/a 1.0 39.8 (16.9-93.3) n/a 1.0 HSA [K573Y]29147.9 (20225.5-42006.2) (2) 182.89 (126.9-263.6) 27.6 (12.8-59.7) ns0.7 (0.3-1.5) (1) Significant difference (p < 0.01) from HSA WT; (2)Significant difference (p < 0.001) from HSA WT; ns = not significantlygreater than wild-type HSA. n/a = not applicable

TABLE 4 mean and range (95% confidence interval) of binding ofFcRn-alexa488 to HSA mutants at pH 5.5 and pH 7.4 pH 5.5 pH 7.4 MeangeoMFI Fold change to WT Mean geoMFI Fold change to WT HSA variant (95%CI) P value (95% CI) (95% CI) P value (95% CI) HSA WT 159.4 (85.6-296.7)n/a 1.0 39.8 (16.9-93.3) n/a 1.0 HSA [K573Y] 29147.9 (20225.5-42006.2)(2, 3, 4, 5) 182.89 (126.9-263.6) 27.6 (12.8-59.7) ns 0.7 (0.3-1.5)HSA[Q522L] 2203.6 (1689.9-2873.3) (2, 4) 13.83 (10.6-18.0) 30.3(10.8-85.1) ns 0.76 (0.3-2.1) HSA[A552V] 644 (464.3-893.4) (1) 4.04(2.9-5.6) 46.1 (36.2-58.7) ns 1.16 (0.9-1.5) HSA[G572R] 6814.2(4453.0-10427.3) (2, 3, 4) 42.76 (27.9-65.4) 31.7 (12.0-84.3) ns 0.8(0.3-2.1) HSA[Q522L, 7572.3 (6586.9-8705.1) (2, 3, 4) 47.51 (41.3-54.6)28.8 (12.5-66.4) ns 0.73 (0.3-1.7) A552V] HSA[Q522L, 28723.1(19498.2-42312.3) (2, 3, 4, 5) 180.23 (122.3-265.5) 37.6 (19.8-71.4) ns0.95 (0.5-1.8) G572R] HSA[A552V, 37739.5 (35785.0-39800.7) (2, 3, 4, 5)236.8 (224.5-249.7) 37.9 (24.3-59.1) ns 0.95 (0.6-1.5) G572R] HSA[Q522L,65180.1 (54138.9-78473.1) (2, 3, 4, 5, 6) 408.98 (339.7-492.4) 46.8(33.6-65.3) ns 1.18 (0.8-1.6) A552V, G572R] (1) Significant difference(p < 0.01) from HSA WT (2) Significant difference (p < 0.001) from HSAWT (3) Significant difference (p < 0.001) from HSA[Q522L] (4)Significant difference (p < 0.001) from HSA[A552V] (5) Significantdifference (p < 0.001) from HSA[G572R] (6) Significant difference (p <0.001) from HSA[K573Y] ns = not significantly greater than wild-type HSAn/a = not applicable

TABLE 5 Mean and range (95% confidence interval) of binding ofFcRn-alexa488 to HSA mutants at pH 5.5 and pH 7.4. pH 5.5 pH 7.4 MeangeoMFI Fold change to WT Mean geoMFI Fold change to WT HSA variant (95%CI) P value (95% CI) (95% CI) P value (95% CI) HSA WT 159.4 (85.6-296.7)n/a 1.0 39.8 (16.9-93.3) ns 1.0 HSA [K573Y] 29147.9 (20225.5-42006.2)(2, 3, 4, 5) 182.89 (126.9-263.6) 27.6 (12.8-59.7) ns 0.7 (0.3-1.5)HSA[Q522L] 2203.6 (1689.9-2873.3) (2, 4) 13.83 (10.6-18.0) 30.3(10.8-85.1) ns 0.76 (0.3-2.1) HSA[A552V] 644 (464.3-893.4) (1) 4.04(2.9-5.6) 46.1 (36.2-58.7) ns 1.16 (0.9-1.5) HSA[G572R] 6814.2(4453.0-10427.3) (2, 3, 4) 42.76 (27.9-65.4) 31.7 (12.0-84.3) ns 0.8(0.3-2.1) HSA[Q522L, 7572.3 (6586.9-8705.1) (2, 3, 4) 47.51 (41.3-54.6)28.8 (12.5-66.4) ns 0.73 (0.3-1.7) A552V] HSA[Q522L, 28723.1(19498.2-42312.3) (2, 3, 4, 5) 180.23 (122.3-265.5) 37.6 (19.8-71.4) ns0.95 (0.5-1.8) G572R] HSA[A552V, 37739.5 (35785.0-39800.7) (2, 3, 4, 5)236.8 (224.5-249.7) 37.9 (24.3-59.1) ns 0.95 (0.6-1.5) G572R] HSA[Q522L,65180.1 (54138.9-78473.1) (2, 3, 4, 5, 408.98 (339.7-492.4) 46.8(33.6-65.3) ns 1.18 (0.8-1.6) A552V, G572R] 7, 8, 9, 10) HSA[Q522L,85950.7 (56455.2-130856.3) (2, 3, 7) 539.3 (354.2-821.1) 28.3(12.6-63.5) ns 0.7 (0.3-1.6) K573Y] HSA[A552V, 92717.2(58348.7-147329.4) (2, 4, 7) 581.8 (366.1-924.4) 36.5 (22.3-59.7) ns 0.9(0.6-1.5) K573Y] HSA[G572R, 42587.6 (28709.3-63174.8) (2, 5) 267.2(180.1-396.4) 30.4 (13.7-67.4) ns 0.8 (0.3-1.7) K573Y] HSA[Q522L,103807.3 (92277.2-116778.1) (2, 3, 4, 7) 651.4 (579.0-732.7) 31.9(16.6-61.2) ns 0.8 (0.4-1.5) A552V, K573Y] HSA[Q522L, 62796.1(40576.1-97184.1) (2, 3, 5, 6) 394.0 (254.6-609.8) 193.9 (166.5-225.7)ns 4.9 (4.2-5.7) G572R, K573Y] HSA[Q522L, 70142.8 (58098.7-84683.9) (2,3, 4, 5, 7) 440.1 (364.6-531.4) 328.9 (303.0-357.0) (1) 8.3 (7.6-9.0)A552V, G572R, K573Y] (1) Significant difference (p < 0.01) from HSA WT;(2) Significant difference (p < 0.001) from HSA WT; (3) Significantdifference (p < 0.001) from HSA[Q5221L]; (4) Significant difference (p <0.001) from HSA[A552V]; (5) Significant difference (p < 0.001) fromHSA[G572R]; (6) Significant difference (p < 0.01) from HSA[K573Y]; (7)Significant difference (p < 0.001) from HSA[K573Y]; (8) Significantdifference (p < 0.001) from HSA[Q522L, A552V]; (9) Significantdifference (p < 0.001) from HSA[Q522L, G572R]; (10) Significantdifference (p < 0.001) from HSA[A552V, G572R]; ns = not significantlygreater than wild-type HSA; n/a = not applicable

TABLE 6 .Rate constants and affinity of soluble HSA mutants tobiotinylated hFcRn/β2M tethered to a Streptavidin sensor chip at acidicpH (pH 6). HSA variant k_(a) (1/Ms) k_(d) (1/s) K_(D) (nM) HSA WT 7.42 ×10⁴ 3.10 × 10⁻² 427 ± 31 HSA [Q522L, A552V, G572R] 3.42 × 10⁴ 1.40 ×10⁻³ 41 ± 1 HSA [K573Y] 2.72 × 10⁴ 1.24 × 10⁻³ 46 ± 1 HSA [A552V/G572R]4.66 × 10⁴ 2.58 × 10⁻³ 55 ± 2 HSA [G572R] 4.66 × 10⁴ 6.21 × 10⁻³ 133 ±1  HSA [Q522L, A552V] 3.73 × 10⁴ 5.00 × 10⁻³ 134 ± 3  HSA [G572K] 4.32 ×10⁴ 6.64 × 10⁻³ 154 ± 4  HSA [Q522F] 3.93 × 10⁴ 8.19 × 10⁻³ 209 ± 8  HSA[G572V] 4.98 × 10⁴ 1.14 × 10⁻² 230 ± 3  HSA [Q522G] 4.81 × 10⁴ 1.27 ×10⁻² 264 ± 10 HSA [Q522M] 1.07 × 10⁴ 3.27 × 10⁻² 305 ± 8  HSA [Q522K]6.07 × 10⁴ 1.82 × 10⁻² 307 ± 24 HSA [G572A] 4.91 × 10⁴ 1.50 × 10⁻² 308 ±17 HSA [Q522Y] 4.66 × 10⁴ 1.46 × 10⁻² 321 ± 32 HSA [A552V] 3.67 × 10⁴1.18 × 10⁻² 322 ± 3  HSA [G572S] 7.24 × 10⁴ 2.29 × 10⁻² 334 ± 39 HSA[Q522I] 5.07 × 10⁴ 1.69 × 10⁻² 338 ± 23 HSA [Q522A] 5.23 × 10⁴ 1.80 ×10⁻² 348 ± 22 HSA [G572H] 6.79 × 10⁴ 2.39 × 10⁻² 356 ± 44 HSA [Q522L]4.07 × 10⁴ 1.26 × 10⁻²  389 ± 194 HSA [Q522V] 1.14 × 10⁴ 1.00 × 10⁻² 880± 44 HSA [Q522W] 2.04 × 10⁴ 3.02 × 10⁻² 1506 ± 197

TABLE 7 .Rate constants and affinity of soluble HSA mutants tobiotinylated hFcRn/β2M tethered to a Streptavidin sensor chip at acidicpH (pH 6). HSA variant k_(a) (1/Ms) k_(d) (1/s) K_(D) (nM) ± SEM HSA WT7.70E+04 3.71E−02 479.4 ± 64.3 HSA [Q522L/A552V/ 7.01E+04 1.76E−03 25.0± 0.6 G572R] HSA [Q522L/A552V] 6.92E+04 6.78E−03 97.9 ± 4.8 HSA[Q522L/G572R] 8.19E+04 4.12E−03 50.2 ± 1.1 HSA [A552V/G572R] 7.63E+043.18E−03 41.7 ± 0.5 HSA [K573Y] 4.79E+04 1.63E−03 33.9 ± 0.6 HSA [G572H]1.07E+05 4.83E−02 452.8 ± 36.7 HSA [G572R] 1.05E+05 1.08E−02 103.1 0.3HSA [G572K] 1.09E+05 1.68E−02 154.4 ± 9.0  HSA [Q522F] 6.37E+04 1.33E−02208.3 ± 3.2  HSA [A552V] 4.38E+04 1.26E−02 286.9 ± 0.3  HSA [Q522I]7.61E+04 2.52E−02 332.1 ± 6.0  HSA [Q522L] 5.78E+04 1.16E−02 201.0 ±1.2  HSA [Q522A] 7.53E+04 3.39E−02 465.0 ± 35.2 HSA [H464Q] NB NB NB NB:No Binding detected

TABLE 8 Binding affinity of soluble mammalian FcRn/β2m binding toimmobilised human HSA variants at acidic (pH 6.0) and neutral (pH 7.3)pH. Analyte pH 6.0⁽¹⁾ pH 7.3⁽²⁾ hsFcRn* cynoFcRn ratFcRn murineFcRnhsFcRn^(ϕ) cynoFcRn ratFcRn murineFcRn Ligand (K_(D) nM, (K_(D) nM,(K_(D) nM, (K_(D) nM, (K_(D) μM, (K_(D) μM, (K_(D) μM, (K_(D) μM, HSAvariant N = 6) N = 4) N = 4) N = 4) N = 4) N = 2) N = 2) N = 2) HSA WT343.9 ± 593.6 ± NB NB NB NB NB NB 36.4 11.9 HSA [K573Y] 18.1 ± 34.7 ±989.2 ± NB >50 45.2 NB NB 1.2 0.7 196.2 HSA[G572R] 58.3 ± 109.1 ± NB NB32.7 ± 21.1 NB NB 2.7 9.5 3.9 HSA[Q522L, 50.1 ± 84.6 ± NB NB >50 33.0 NBNB A552V] 1.6 5.1 HSA[Q522L, 27.8 ± 49.5 ± 4754.1 ± NB 18.0 ± 10.7 NB NBG572R] 0.6 0.7 354.7 1.0 HSA[A552V, 27.1 ± 48.4 ± 4823.3 ± NB 20.5 ±13.2 NB NB G572R] 0.7 0.8 307.3 0.4 HSA[Q522L, 16.9 ± 24.5 ± 2245.5 ± NB16.1 ± 7.6 NB NB A552V, G572R] 0.5 0.2 119.1 1.6 ⁽¹⁾Mean ± SEM valuesindicated in nanomolar calculated from 1:1 binding model. ⁽²⁾HumanFcRn-β2m data (hsFcRn) are indicated as Mean ± SEM; cyno FcRn-β2mrepresent the average of two experimental replicates. KD values werecalculated from sensogram data fit to a 1:1 steady-state model *Humansoluble FcRns (hsFcRn); NB, Non-binder.

TABLE 9 Serum concentration (μg/mL; mean ± SEM) of HSA variants intransgenic mice expressing human FcRn receptor HSA [Q522L, HSA [Q522L,Days HSA WT A552V] A552V, G572R] HSA [573Y] 0 0.042 0.25 1 2 3 4 5 7 1621 29

TABLE 10 Pharmacokinetics analysis of the serum concentration of HSAvariants in transgenic mice expressing human FcRn receptor. Area Underthe Curve HSA variant AUC (μg/mL*d) SD N p value HSA WT 344.6 18.2 3 HSA[K573Y] 574.8 30 3 <0.0001 HSA [Q522L, A552V] 597 26.2 3 <0.0001 HSA[Q522L, A552V, G572R] 618 29 3 <0.0001 Mean Residence Time HSA variantMRT (days) SD N p value HSA WT 3.31 0.08 3 HSA [K573Y] 4.83 0.15 3<0.0001 HSA [Q522L, A552V] 5.06 0.12 3 <0.0001 HSA [Q522L, A552V, G572R]5.02 0.12 3 <0.0001 Clearance HSA variant Cl (ml/kg*d) SD N p value HSAWT 29.1 1.5 3 HSA [K573Y] 17.4 0.9 3 <0.0001 HSA [Q522L, A552V] 16.8 0.73 <0.0001 HSA [Q522L, A552V, G572R] 16.2 0.8 3 <0.0001 p valuesrepresent ANOVA analysis of difference between the selected HSA variantand WT

The invention claimed is:
 1. A recombinant serum albumin variant, orfunctional fragment thereof, comprising an amino acid substitutionselected from the group consisting of: (i) an amino acid selected fromthe group consisting of glycine, isoleucine, lysine, methionine,phenylalanine, tryptophan, tyrosine, valine and leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,and valine substituted for alanine at position 552 of SEQ ID NO: 1; or(ii) an amino acid selected from the group consisting of glycine,isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine,valine and leucine substituted for glutamine at a position correspondingto amino acid 522 of SEQ ID NO: 1, and an amino acid selected from thegroup consisting of alanine, glutamic acid, histidine, serine, lysineand arginine substituted for glycine at position 572 of SEQ ID NO: 1; or(iii) valine substituted for alanine at a position corresponding toamino acid 552 of SEQ ID NO: 1, and an amino acid selected from thegroup consisting of alanine, glutamic acid, histidine, serine, lysineand arginine substituted for glycine at position 572 of SEQ ID NO: 1;and (iv) an amino acid selected from the group consisting of glycine,isoleucine, lysine, methionine, phenylalanine, tryptophan, tyrosine,valine and leucine substituted for glutamine at a position correspondingto amino acid 522 of SEQ ID NO: 1, valine substituted for alanine atposition 552 of SEQ ID NO: 1, and an amino acid selected from the groupconsisting of alanine, glutamic acid, histidine, serine, lysine andarginine substituted for glycine at position 572 of SEQ ID NO:
 1. 2. Therecombinant serum albumin variant, or functional fragment thereof, ofclaim 1, wherein the recombinant serum albumin variant, or functionalfragment thereof, binds with increased affinity to FcRn compared to aserum albumin set forth in SEQ ID NO: 1 or wherein the recombinant serumalbumin variant, or functional fragment thereof, has an increased serumhalf-life compared to a serum albumin set forth in SEQ ID NO:
 1. 3. Therecombinant serum albumin variant, or functional fragment thereof, ofclaim 2, wherein the binding affinity is measured at acidic pH.
 4. Therecombinant serum albumin variant, or functional fragment thereof,according to claim 1, further comprising tyrosine substituted for lysineat a position corresponding to amino acid 573 of SEQ ID NO:
 1. 5. Therecombinant serum albumin variant, or functional fragment thereof,according to claim 1, comprising: (i) leucine substituted for glutamineat a position corresponding to amino acid 522 of SEQ ID NO: 1; and/or(ii) valine substituted for alanine at a position corresponding to aminoacid 552 of SEQ ID NO: 1; and/or (iii) arginine substituted for glycineat a position corresponding to amino acid 572 of SEQ ID NO:
 1. 6. Therecombinant serum albumin variant, or functional fragment thereof,according to claim 1, comprising: (i) leucine substituted for glutamineat a position corresponding to amino acid 522 of SEQ ID NO: 1, andarginine substituted for glycine at a position corresponding to aminoacid 572 of SEQ ID NO: 1; or (ii) leucine substituted for glutamine at aposition corresponding to amino acid 522 of SEQ ID NO: 1, and valinesubstituted for alanine at a position corresponding to amino acid 552 ofSEQ ID NO: 1; or (iii) valine substituted for alanine at a positioncorresponding to amino acid 552 of SEQ ID NO: 1, and argininesubstituted for glycine at a position corresponding to amino acid 572 ofSEQ ID NO: 1; or (iv) leucine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1, and tyrosinesubstituted for lysine at a position corresponding to amino acid 573 ofSEQ ID NO: 1; or (v) valine substituted for alanine at a positioncorresponding to amino acid 552 of SEQ ID NO: 1, and tyrosinesubstituted for lysine at a position corresponding to amino acid 573 ofSEQ ID NO: 1; or (vi) arginine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1, and tyrosinesubstituted for lysine at a position corresponding to amino acid 573 ofSEQ ID NO: 1; or (vii) leucine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1, valine substituted foralanine at a position corresponding to amino acid 552 of SEQ ID NO: 1,and arginine substituted for glycine at a position corresponding toamino acid 572 of SEQ ID NO: 1; or (viii) leucine substituted forglutamine at a position corresponding to amino acid 522 of SEQ ID NO: 1,valine substituted for alanine at a position corresponding to amino acid552 of SEQ ID NO: 1, and tyrosine substituted for lysine at a positioncorresponding to amino acid 573 of SEQ ID NO: 1; or (ix) leucinesubstituted for glutamine at a position corresponding to amino acid 522of SEQ ID NO: 1, arginine substituted for glycine at a positioncorresponding to amino acid 572 of SEQ ID NO: 1, and tyrosinesubstituted for lysine at a position corresponding to amino acid 573 ofSEQ ID NO: 1; or (x) leucine substituted for glutamine at a positioncorresponding to amino acid 522 of SEQ ID NO: 1, valine substituted foralanine at position 552 of SEQ ID NO: 1, arginine substituted forglycine at a position corresponding to amino acid 572 of SEQ ID NO: 1,and tyrosine substituted for lysine at a position corresponding to aminoacid 573 of SEQ ID NO: 1.